Extracellular Matrix for Treating Pelvic Floor Disorders and Skeletal Muscle Degeneration

ABSTRACT

Described herein are compositions comprising decellularized extracellular matrix derived from skeletal muscle or other suitable tissue, and therapeutic uses thereof. Methods for treating, repairing or regenerating defective, diseased, damage, ischemic, ulcer cells, tissues or organs in a subject preferably a human, with diseases associated with muscular degeneration, using a decellularized extracellular matrix of the invention are provided. Methods of preparing culture surfaces and culturing cells with absorbed decellularized extracellular matrix are provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of U.S. Provisional PatentApplication No. 62/507,850, filed May 18, 2017, the entire contents ofwhich is incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.R01HL113468 awarded by National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The extracellular matrix (ECM) of each tissue contains similarcomponents; however, each individual tissue is composed of a uniquecombination of proteins and proteoglycans (Lutolf and Hubbell, 2005;Uriel et al., 2009). Recent studies have shown that the ECM of varioustissues can be isolated through decellularization and utilized as atissue engineering scaffold (Merritt et al.; Ott et al., 2008; Singelynet al., 2009; Uygun et al., 2010; Valentin et al., 2010; Young et al.,2011). Other decellularized ECM materials have been used for a varietyof applications for tissue repair (Crapo et al., 2011; Gilbert et al.,2006). These scaffolds are known to promote cellular influx in a varietyof tissues (Numata et al., 2004; Rieder et al., 2006). Their degradationproducts have angiogenic (Li et al., 2004) and chemoattractant (Badylaket al., 2001; Beattie et al., 2008; Li et al., 2004; Zantop et al.,2006) properties, and also promote cell migration and proliferation(Reing et al., 2009). After removal of the cellular antigens, thesescaffolds are considered biocompatible, and both allogeneic andxenogeneic ECM devices have been approved by the FDA and are in clinicaluse (Badylak, 2007).

Hydrogels derived from decellularized ECMs, including myocardium(Singelyn et al., 2009), pericardium (Seif-Naraghi et al., 2010), andadipose tissue (Young et al., 2011), were recently developed whichassemble into porous and fibrous scaffolds upon injection in vivo. Ithas been shown that the injectable hydrogel derived from ventricular ECMpromoted endogenous cardiomyocyte survival and preserved cardiacfunction post-myocardial infarction (Singelyn et al., 2012). ECMhydrogels have also been produced for treatment of ischemic muscletissue due to peripheral artery disease and critical limb ischemia (SeePCT/US2012/054058).

A liquid form of skeletal muscle matrix was shown to promote thedifferentiation and maturation of C2C12 skeletal myoblast progenitorswhen used as a cell culture coating due to its ability to retain acomplex mixture of skeletal muscle ECM proteins, peptides, andproteoglycans (DeQuach et al., 2010). A decellularized skeletal musclescaffold has been previously explored for replacement of a muscle defect(Merritt et al.; Wolf et al., 2012), yet this intact scaffold would notbe amenable to treating certain non-skeletal muscle tissue disease, suchas the peripheral artery disease (PAD) and CLI.

Skeletal muscles are composed of bundles of highly oriented and densemuscle fibers, each a multinucleated cell derived from myoblasts. Themuscle fibers in native skeletal muscle are closely packed together inan extracellular three-dimensional matrix to form an organized tissuewith high cell density and cellular orientation to generate longitudinalcontraction. Skeletal muscle can become dysfunctional due to a varietyof different factors including trauma, atrophy or degeneration.

The reconstruction of skeletal muscle, which is lost by injury, tumorresection, or various myopathies, is limited by the lack of functionalsubstitutes. Surgical treatments, such as muscle transplantation andtransposition techniques, have had some success; however, there stillexists a need for alternative therapies. Tissue engineering approachesoffer potential new solutions; however, current options offer incompleteregeneration. Many naturally derived as well as synthetic materials havebeen explored as scaffolds for skeletal tissue engineering, but noneoffer a complex mimic of the native skeletal extracellular matrix, whichpossesses important cues for cell survival, differentiation, andmigration.

The extracellular matrix consists of a complex tissue-specific networkof proteins and polysaccharides, which help regulate cell growth,survival and differentiation. Despite the complex nature of native ECM,in vitro cell studies traditionally assess cell behavior on single ECMcomponent coatings, thus posing limitations on translating findings fromin vitro cell studies to the in vivo setting. Overcoming this limitationis important for cell-mediated therapies, which rely on cultured andexpanded cells retaining native cell behavior over time.

Typically, purified matrix proteins from various animal sources areadsorbed to cell culture substrates to provide a protein substrate forcell attachment and to modify cellular behavior. However, theseapproaches would not provide an accurate representation of the complexmicroenvironment. More complex coatings have been used, such as acombination of single proteins, and while these combinatorial signalshave shown to affect cell behavior, it is not as complete as in vivo.For a more natural matrix, cell-derived matrices have been used.Matrigel is a complex system; however, it is derived from mouse sarcoma,and does not mimic any natural tissue. While many components of ECM aresimilar, each tissue or organ has a unique composition, and a tissuespecific naturally derived source may prove to be a better mimic of thecell microenvironment.

Pelvic Floor Disorders (PFD)

Pelvic floor disorders (PFD), which include urinary (UI) and fecalincontinence (FI), and pelvic organ prolapse (POP), are debilitatingconditions that affect a quarter of the U.S. female population. Theprevalence of UI, which is the most common PFD, is 17%, followed by Hwith 9-15% prevalence, and POP with 3% prevalence rate. By 2050, thenumber of women with PFD is predicted to increase to 43.8 million.Maternal birth trauma and consequent dysfunction of urethral sphincter(US), external anal sphincter (EAS) and pelvic floor (PFM) skeletalmuscles, is a leading risk factor for UI, H, and POP, respectively.Currently, there are no preventive measures, beyond Cesarean section,and the existing treatments are associated with significant morbidities,while offering marginal promise at best.

Focal Skeletal Muscle Degeneration

Dysfunction of striated muscles, which include the rotator cuff muscles(supraspinatus, infraspinatus, teres minor, subcapularis), hip abductormuscle (gluteus medius, gluteus minimus, gluteus maximus, and shortexternal rotators), foot and ankle muscles (tibialis posterior,gastrocnemius, soleus), lumbar spine muscles (multifidus, erectorspinae), and knee extensor muscles (quadriceps), all suffer from fattyatrophy and muscle degeneration as a consequence of chronic jointdisease and other neuromuscular pathologies. This loss of muscleinterferes with muscle and joint function, which negatively impactsquality of life.

SUMMARY OF THE INVENTION

The primary therapeutic goal in female pelvic medicine is to restorenormal pelvic floor function. Despite this, the current standardtreatments are compensatory, as they do not directly target sphinctericand supportive muscle dysfunction and do not reverse the existing injuryor halt functional deterioration. In contrast, the present inventionprovides a novel injectable biomaterial scaffold, derived fromdecellularized skeletal muscle extracellular matrix (ECM), whichcapitalizes on the endogenous regenerative potential of the host tissueand bridges this therapeutic void by restoring and preserving functionof injured pelvic muscles and striated focal skeletal muscles.

In embodiments the invention provides an injectable biomaterial scaffoldand a minimally invasive delivery system for the treatment of US, EAS,and PFM. Upon injection, the material will set up into a porous andfibrous scaffold that will facilitate endogenous cell infiltration toregenerate and heal the damaged muscles post-vaginal delivery therebypreventing the development of PFD.

The present invention provides a new use for ECM materials that havealready shown to promote differentiation of muscle progenitors in vitro(DeQuach et al., PLoS One, 2010), as well as migration of muscleprogenitors, decrease in cell death, and increase in neovascularizationand muscle development in vivo in hindlimb ischemia models of muscledamage (DeQuach et al., ECM, 2012; Ungerleider, et al., JACC: BTS,2016). This approach has significant clinical application as injectablescaffolds can be delivered with minimal invasiveness, thereby enablingtheir administration in labor and delivery units and outpatientsettings, and reducing patient recovery time and perioperative morbiditycompared to surgical approaches. This biomaterials based approach isalso likely to reach the clinic sooner as it is devoid of the hurdlesrelated to stem-cell based therapies and obviates the difficulties andexpense associated with administration of exogenous growth factors. Infact, the analogous hydrogel derived from decellularized porcinemyocardium is already in clinical trials for myocardial repair and istwo orders of magnitude cheaper to manufacture compared to cellproducts. In addition, stem cell based therapies suffer from poor cellsurvival and have led to increased connective tissue rather than muscleregeneration in urethral sphincter (Sadeghi, et al. Int Urogynecol J,2016).

Importantly, application of the injectable skeletal muscle matrixscaffold to treat pelvic muscles, injured during vaginal delivery, canshift the current clinical paradigm towards prevention of postpartummuscle dysfunction, which is essential for meaningful advances to occurin female pelvic medicine. Ultimately, this innovative approach willreduce the incidence of PFD and improve the lives of millions of women.

Presently, postpartum pelvic floor muscle dysfunction is thought to bedue to radiologically detected muscle tears or avulsions, which havebeen the primary focus of the literature to date. Morgan D M, Larson K,Lewicky-Gaupp C, Fenner D E, DeLancey J O 2011 Vaginal support asdetermined by levator ani defect status 6 weeks after primary surgeryfor pelvic organ prolapse. Int J Gynaecol Obstet 114:141-144; Kearney R,Fitzpatrick M, Brennan S, Behan M, Miller J, Keane D, O'Herlihy C,DeLancey J O L 2010 Levator ani injury in primiparous women with forcepsdelivery for fetal distress, forceps for second stage arrest, andspontaneous delivery. International journal of gynaecology andobstetrics 111:19-22; DeLancey J O, Kearney R, Chou Q, Speights S, BinnoS 2003 The appearance of levator ani muscle abnormalities in magneticresonance images after vaginal delivery. Obstet Gynecol 101:46-53; DietzH P, Lanzarone V 2005 Levator trauma after vaginal delivery. ObstetGynecol 106:707-712.

The present invention provides that direct measures of pelvic floormuscle properties indicate that strains imposed on the pelvic floormuscles (PFMs) during simulated birth injury (SBI) result in acutesarcomere hyperelongation and myofibrillar disruption, not avulsions(FIGS. 13A-13B and 14A-14B). These acute events lead to PFM fibrosis,similar to the degenerative changes observed in human PFMs, as well asfunctional alterations. However, the population of resident muscleprogenitors (satellite cells) appears to be normal. This suggests thatviable cells are present in PFMs, but are not able to mount a responsesufficient for regeneration after birth injury. One would not expect tobe able to repair what was believed to be a torn muscle (i.e., anavulsion) with an injectable ECM hydrogel material.

The existing knowledge regarding the impact of aging on PFMs is mainlyderived from conventional radiological examinations that are compromisedby low resolution. These imaging studies use volumetric measures, suchas muscle cross-sectional area, and have failed to identify age-relatedatrophy in PFMs, due to their inability to distinguish between, andidentify changes in, contractile vs. ECM components. Morris V C, MurrayM P, Delancey J O, Ashton-Miller J A 2012 A comparison of the effect ofage on levator ani and obturator internus muscle cross-sectional areasand volumes in nulliparous women. Neurourol Urodyn 31:481-486.

Employing experimental tools, not previously utilized in female pelvicmedicine, the present invention determined that physiologicalalterations, such as decrease in force production and excursion, as wellas degenerative changes, such as fibrosis, occur in aged PFMs. Thesenovel tissue-level findings provide a mechanistic link between aging andPFM dysfunction and serve as an impetus for providing preventativestrategies to mitigate the untoward aging effects. Furthermore,identification of substantial fibrosis in aged PFMs provides noveltreatment approaches, aimed at reversing functionally relevantpathological changes by promoting muscle regeneration. Alperin M, Cook MS, Tuttle L I, Esparza M, Lieber R L. Impact of vaginal parity and agingon the architectural design of pelvic floor muscles. Am J ObstetGynecol. 2016 September; 215(3):312.e.1-9. PAM: 26953079 PMCID: 5003683;Cook M S, Bou-Malham L, Esparza M C, Alperin M. Age-related alterationsin obturator internus muscle. Int Urogynecol J. 2016 Oct. 4. [Epub aheadof print] MUD: 27704154.

Furthermore, urinary and anal sphincters are thin circular muscles,which are very different types of muscles than previously targeted forECM hydrogel or patch administration.

The present invention provides biomaterials comprising extracellularmatrix (ECM) derived from skeletal muscle or other suitable tissue, andmethod of use thereof, for therapeutic treatment of skeletal muscledegeneration. The invention provides for the treatment of acutesarcomere hyperelongation and myofibrillar disruption, rather thanavulsions. The invention provides for the treatment of PFM fibrosis andother pelvic floor disorders (PFD), which include urinary (UI) and fecalincontinence (FI), and pelvic organ prolapse (POP), and/or othermuscular diseases. In embodiments, the targeted muscle tissue includesurinary and anal sphincters. The invention provides for the treatment ofdysfunction of pelvic striated muscles, which include external urethral(EUS) and external anal (EAS) sphincteric muscles, and pelvic floormuscles (PFM). The invention provides for the treatment of patients withRectal Prolapse (RP), Stress Urinary Incontinence (SUI), Mixed UrinaryIncontinence (MUI), SUI/MUI, patients with SUI/MUI and intrinsicsphincter deficiency, patients with SUI/MUI and pelvic floor muscle(PFM) dysfunction, and vaginally parous women.

With respect to orthopedic application, the invention provides for thetreatment of degenerative muscle conditions which are also not avascularischemic type injuries. Here there is accelerated apoptosis in suchtissues, and the prior art has attempted to replace cells as atreatment. The present invention provides, however, a unique pathologyshowing that a skeletal muscle ECM hydrogel can improve muscledegeneration.

In certain embodiments, the present invention provides injectablebiomaterials comprising skeletal muscle extracellular matrix, and methodof use thereof, for treating orthopedic diseases, and symptoms andassociated complications with these diseases. Orthopedic conditionsresulting from skeletal muscle degenerative disease contemplated fortreatment by the present invention include dysfunction of striatedmuscles, which include the rotator cuff muscles (supraspinatus,infraspinatus, teres minor, subcapularis), hip abductor muscle (gluteusmedius, gluteus minimus, gluteus maximus, and short external rotators),foot and ankle muscles (tibialis posterior, gastrocnemius, soleus),lumbar spine muscles (multifidus, erector spinae), and knee extensormuscles (quadriceps). Patients in need may suffer from fatty atrophy andmuscle degeneration as a consequence of chronic joint disease and otherneuromuscular pathologies. This loss of muscle interferes with muscleand joint function, which negatively impacts quality of life. Theinvention provides for the treatment of shoulder, hip, foot and ankle,lumbar spine, and knee degenerative joint and tendon diseases, which areassociated with focal muscle atrophy and degeneration. Patients in needinclude those with tendinopathy and/or tendon rupture, patients withosteoarthritis, patients with rheumatoid arthritis, and patients withlower back pain for example.

In certain embodiments, the present invention provides compositions andmethods comprising injecting or implanting in a subject in need aneffective amount of a composition comprising decellularizedextracellular matrix derived from skeletal muscle tissue. In otherembodiments, the present invention provides a method comprisinginjecting or implanting in a subject in need a composition comprisingdecellularized extracellular matrix derived from a suitable tissue,including but not limited to, cardiac, pericardial, liver, brain, smallintestine submucosa, bladder, and vascular tissue. In certainembodiments, the injection or implantation of said composition repairsdamage to skeletal muscle tissue sustained by said subject. In otherembodiments, the injection or implantation of said composition repairsdamage caused by muscular degeneration in said subject.

The composition of the present invention comprising the ECM material candegrade within about one month, two months, or three months followinginjection or implantation. In certain embodiments, the injection orimplantation of said composition repairs damage to skeletal muscletissue sustained by said subject. In certain embodiments, the injectionor implantation of said composition repairs damage caused by musculardegeneration in said subject. Herein, said effective amount can be anamount that increases blood flow in the area of the injection orimplantation or treated limb of the treated subject. In some instances,the effective amount is an amount that induces new vascular formation inthe area of the injection or implantation of the treated subject. Thepresent invention provides that a liquid form of skeletal muscle matrixcan assemble into a fibrous scaffold upon injection in vivo. Thematerial can also be processed into a lyophilized form that onlyrequires sterile water, PBS, or saline to re-suspend prior to injection,which can provide ease of storage and use in a clinical setting. Thecomposition can further comprise cells, drugs, proteins, orpolysaccharides. In some instances, the composition is coated on adevice such as an implant. The composition can be delivered as a liquid,and in many instances, the composition can transition to a gel formafter delivery. In certain embodiments, the composition is delivered asa powder.

In one aspect, the invention provides a composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissueor other suitable tissues. The composition can be injectable. Thecomposition can be formulated into a powder or particulate. In otherinstances, the composition can be formulated to be in liquid form atroom temperature, typically 20° C. to 25° C., and in gel form at atemperature greater than room temperature or greater than 35° C. In someinstances, the composition is configured to be delivered to a tissueparenterally, such as through a small gauge needle (e.g., 27 gauge orsmaller). In some instances, said composition is suitable for directimplantation into a patient. The composition can be formulated either ina dry or hydrated form to be placed on or in wounds.

In some instances the composition comprises native proteins. In someinstances the composition comprises native peptides. In some instancesthe composition comprises native glycosaminoglycans. In some instances,the composition further comprises non-naturally occurring factors thatrecruit cells into the composition, encourage growth or preventinfection. In some embodiments, the composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissueherein retains native glycosaminoglycans. In some instances, thecomposition comprises naturally occurring factors that recruit cellsinto the composition, encourage growth or prevent infection.

In some instances, the composition further comprises a population ofexogenous or autologous therapeutic cells. The cells can be stem cellsor other precursors of skeletal muscle cells or other cell types.

In some instances, the composition further comprises a therapeuticagent, and as such is configured as a drug delivery vehicle. In someinstances, the composition is configured to coat surfaces, such astissue culture plates or scaffolds, to culture skeletal muscle, skeletalmuscle cells, or other cell types relevant to skeletal muscle repair.

In an aspect, a method of producing a composition is disclosed hereinthat comprises decellularized skeletal muscle or other tissueextracellular matrix comprising: obtaining from a subject a skeletalmuscle or other suitable tissue sample having an extracellular matrixand non-extracellular matrix components; processing skeletal muscle orother tissue sample to remove the non-extracellular matrix component toobtain decellularized skeletal muscle or other tissue extracellularmatrix and extracellular proteins and polysaccharides; and sterilizingthe decellularized skeletal muscle or other tissue extracellular matrix.In some instances, said method is performed aseptically withoutsterilization. In some instances, said method further comprises the stepof lyophilizing and grinding up the decellularized skeletal muscle orother tissue extracellular matrix. In some instances, said methodfurther comprises the step of enzymatically treating, solubilizing, orsuspending the decellularized skeletal muscle or other tissueextracellular matrix. In some instances, said decellularized skeletalmuscle or other tissue extracellular matrix is digested with pepsin at alow pH.

In some instances, said method further comprises the step of suspendingand neutralizing said decellularized skeletal muscle or other tissueextracellular matrix in a solution. In some instances, said solution isa phosphate buffered solution (PBS) or saline solution which can beinjected through a high gauge needle into the desired tissue or organ.In some instances, said composition is formed into a gel at bodytemperature. In some instances, said composition further comprisescells, drugs, proteins or other therapeutic agents that can be deliveredwithin or attached to the composition before, during or after gelation.

In some instances, said solution is placed into tissue culture plates orwells, incubated at above 35° C. or about 37° C. to form into a gel thatis used for cell culture. In one aspect, the invention provides a methodof culturing cells on an adsorbed matrix comprising the steps of:providing a solution comprising decellularized extracellular matrixderived from skeletal muscle or other suitable tissues into a tissueculture device; incubating said tissue culture plates device; removingsaid solution; and culturing cells on the adsorbed matrix. In someinstances, said cells are skeletal muscle cells or other cell typesrelevant to skeletal muscle or other tissue repair.

In an aspect, a method of culturing cells on an adsorbed matrixcomprises the steps of: providing a solution comprising decellularizedextracellular matrix derived from skeletal muscle or other suitabletissues into a tissue culture device; incubating said tissue cultureplates device; removing said solution; and culturing cells on theadsorbed matrix. In some instances, said cells are skeletal myoblasts,stem cells or other cell types relevant to skeletal muscle repair.

In one aspect, the invention provides a therapeutic method for skeletalmuscle and/or other tissue (such as ischemic tissue, or ulcer tissue)repair in a subject comprising injecting or implanting a therapeuticallyeffective amount of a composition comprising decellularizedextracellular matrix derived from skeletal muscle or other suitabletissue into a subject in need thereof. In an aspect, a therapeuticmethod for skeletal muscle or other tissue repair in a subject comprisesimplanting a composition comprising decellularized extracellular matrixderived from skeletal muscle or other suitable tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F illustrate decellularization and tissue processing. FIG. 1Ashows decellularized skeletal muscle matrix. FIG. 1B shows lyophilizedskeletal muscle matrix prior to milling. FIG. 1C shows digested skeletalmuscle matrix. FIG. 1D shows in vitro gel of the skeletal muscle matrixwith media on top in right well. FIG. 1E shows that skeletal musclematrix that has been digested and re-lyophilized FIG. 1F showsre-lyophilized skeletal muscle matrix resuspended using only sterilewater.

FIGS. 2A-2B illustrate in vitro mitogenic activity assay. FIG. 2A showsrat aortic smooth muscle cells and FIG. 2B shows that C2C12 skeletalmyoblasts were cultured using growth media with the addition of degradedskeletal muscle matrix, collagen, or pepsin. Proliferation rate wasincreased for both cell types when cultured in the presence of skeletalmuscle matrix degradation products.

FIG. 3 illustrates Rheological data. A representative trace of thestorage (G′) and loss (G″) moduli for the skeletal muscle matrix gel isshown.

FIGS. 4A-4C illustrate skeletal muscle matrix delivery and gelation insitu. FIG. 4A shows intramuscular injection of the skeletal musclematrix material. FIG. 4B shows gelation of the skeletal muscle matrix insitu after 20 minutes as seen after excision of the muscle; arrowdenotes the white matrix. FIG. 4C shows DAB staining of thebiotin-labeled skeletal muscle matrix that gelled within the muscle.Scale bar at 200 μm.

FIGS. 5A-5B illustrate scanning electron microscopy. Micrograph of across-section of skeletal muscle matrix formed FIG. 5A in vitro, andFIG. 5B 20 minutes post-subcutaneous injection. Note the formation ofthe assembled fibers on the nano- and micro-scale. Scale bar at 100 μm.

FIGS. 6A-6D illustrate quantification of arterioles. FIG. 6A showscollagen and FIG. 6B shows skeletal muscle matrix injection regionsstained with anti-alpha-SMA (red greyscale) to determine arterioleformation. Vessels with a clear lumen are seen within the injectionregion at 5 days. Scale bar at 100 μm. Quantification of the vesseldensity at 3, 5, 7, and 14 days for vessels with a lumen FIG. 6C >10 μmor FIG. 6D >25 μm demonstrated that the skeletal muscle matrix increasedneovascularization. Vessels were, on average, larger in the skeletalmuscle matrix when compared to collagen.

FIGS. 7A-7C illustrates quantification of endothelial cell recruitment.FIG. 7A shows collagen and FIG. 7B shows skeletal muscle matrixinjection regions stained with isolectin (green greyscale) to assessendothelial cell infiltration at 5 days. Scale bar at 100 μm. * anddotted line denote area of material. FIG. 7C shows that endothelial cellinfiltration at 3, 5, 7, and 14 days was similar across all four timepoints, but was significantly greater in the skeletal muscle matrixinjection region at 3 and 7 days post-injection.

FIGS. 8A-8D illustrates proliferating muscle cell recruitment. FIG. 8Ashows collagen injection region and FIG. 8B shows skeletal muscle matrixinjection region at 5 days with desmin-stained cells (green greyscale)co-labeled with Ki67 (red greyscale). Arrows denote desmin and Ki67positive cells. Scale bar at 20 μm. Insert shows positive desminstaining of healthy skeletal muscle, scale bar at 100 μm. FIG. 8C showsquantification of desmin-positive cells in the skeletal muscle matrixcompared to collagen normalized to area. Note that there aresignificantly more desmin-positive cells in the skeletal muscle matrix.FIG. 8D shows that, of these desmin-positive cells, a majority of thecells are proliferating as seen by Ki67 co-labeling.

FIGS. 9A-9C illustrate muscle progenitor infiltration. MyoD positivecells (green greyscale) in FIG. 9A collagen and FIG. 9B skeletal musclematrix injection regions at 5 days. Area of injection is denoted by thedotted line. Scale bar at 20 μm. FIG. 9C shows graph of MyoD-positivecells normalized to the area for the injection region. The number ofMyoD-positive cells was significantly higher in the skeletal musclematrix regions at all time points.

FIGS. 10A-10B illustrate skeletal muscle matrix delivery and gelation insitu. FIG. 10A shows a gross image of rabbit supraspinatus musclefollowing intramuscular injection of the skeletal muscle matrixmaterial. FIG. 10B shows Hematoxylin and Eosin staining of rabbitsupraspinatus muscle cross sections demonstrating the gelation of thegelation of the skeletal muscle matrix in situ. Arrows denote the gelledwhite matrix within the muscle. Scale bar at 500 μm.

FIGS. 11A-11B illustrate skeletal muscle matrix delivery and gelation inrat pelvic floor. FIG. 11A shows a proximal section of iliococcygeusmuscle following intramuscular injection of the skeletal muscle matrixmaterial. FIG. 11B shows a proximal section of pubococcygeus followingintramuscular injection of the skeletal muscle matrix material. Arrowsdenote the gelled white matrix prelabeled with Alexa Fluor 568. Green(laminin) Scale bar at 500 μm.

FIG. 12 illustrates skeletal muscle matrix delivery and gelation in ratexternal urethral sphincter. Arrows denote the gelled white matrixprelabeled with Alexa Fluor 568. Green (laminin), Blue (cell nuclei).Scale bar at 100 μm.

FIGS. 13A-13B illustrate transmission electron microscopy images ofexternal urethral sphincter longitudinal sections. FIG. 13A showsuninjured control. FIG. 13B shows the departure from normal muscleappearance following simulated birth injury, indicated by misalignmentand smearing of sarcomeres. Scale bar at 1 μm.

FIGS. 14A-14B illustrate transmission electron microscopy images ofpubococcygeus longitudinal sections. FIG. 14A shows uninjured control.FIG. 14B shows the departure from normal muscle appearance followingsimulated birth injury, indicated by misalignment and smearing ofsarcomeres. Scale bar at 2 μm. Adapted from Catanzarite, et al. AM JObstet Gynecol (2018). 218:5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a decellularized skeletal muscleextracellular matrix (ECM) composition, and method of use thereof, forpreventing and treating muscle degeneration and other tissue damage, aswell as for restoring muscle mass and function in certain diseases.Described herein are compositions comprising ECM derived from skeletalmuscle tissue or other suitable tissues, including, but not limited to,cardiac, pericardial, liver, brain, small intestine submucosa, bladder,and vascular tissue, which can be used for injection into skeletalmuscle tissue and/or other tissues in need of therapeutic treatment. Incertain embodiments, the ECM composition of the present invention canalso be used to support injured tissue or change the mechanicalproperties. In certain embodiments, the ECM composition as describedherein can help regenerate defective or absent skeletal muscle andrestore muscle mass and function. In certain embodiments, the injectionor implantation of said composition repairs damage to skeletal muscletissue sustained by said subject.

In certain embodiments, the present invention provides a decellularizedskeletal muscle extracellular matrix (ECM) composition, and method ofuse thereof, for treating acute sarcomere hyperelongation andmyofibrillar disruption. The invention provides for the treatment of PFMfibrosis and other pelvic floor disorders (PFD), which include urinary(UI) and fecal incontinence (FI), and pelvic organ prolapse (POP),and/or other muscular diseases. In embodiments, the targeted muscletissue includes urinary and anal sphincters. The invention provides forthe treatment of dysfunction of pelvic striated muscles, which includeexternal urethral (EUS) and external anal (EAS) sphincteric muscles, andpelvic floor muscles (PFM). The invention provides for the treatment ofpatients with Rectal Prolapse (RP), Stress Urinary Incontinence (SUI),Mixed Urinary Incontinence (MUI), SUI/MUI, patients with SUI/MUI andintrinsic sphincter deficiency, patients with SUI/MUI and pelvic floormuscle (PFM) dysfunction, and vaginally parous women.

The invention provides for the prevention and treatment of degenerativemuscle conditions associated with orthopedic disease. In certainembodiments, the present invention provides therapeutic biomaterialscomprising skeletal muscle extracellular matrix, and method of usethereof, for treating orthopedic diseases, and symptoms and associatedcomplications with these diseases. Orthopedic conditions resulting fromskeletal muscle degenerative disease contemplated for treatment by thepresent invention include dysfunction of striated muscles, which includethe rotator cuff muscles (supraspinatus, infraspinatus, teres minor,subcapularis), hip abductor muscle (gluteus medius, gluteus minimus,gluteus maximus, and short external rotators), foot and ankle muscles(tibialis posterior, gastrocnemius, soleus), lumbar spine muscles(multifidus, erector spinae), and knee extensor muscles (quadriceps).Patients in need may suffer from fatty atrophy and muscle degenerationas a consequence of chronic joint disease and other neuromuscularpathologies. This loss of muscle interferes with muscle and jointfunction, which negatively impacts quality of life. The inventionprovides for the treatment of shoulder, hip, foot and ankle, lumbarspine, and knee degenerative joint and tendon diseases, which areassociated with focal muscle atrophy and degeneration. Patients in needinclude those with tendinopathy and/or tendon rupture, patients withosteoarthritis, patients with rheumatoid arthritis, and patients withlower back pain for example.

The present invention further provides a method of delivering thedecellularized skeletal muscle extracellular matrix (ECM) composition ofthe present invention, with or without other therapeutic agents,including cells, into one or more injured tissues or organs damaged bycertain disease conditions or trauma. In some instances, methods ofdelivery are described wherein the skeletal muscle ECM composition ofthe present invention can be placed in contact with a defective,diseased or absent muscle tissues or other injured tissues, resulting inskeletal muscle tissue regeneration and restoration of muscle mass andfunction. Exemplary methods for delivery of a composition comprising theskeletal muscle ECM include, but are not limited to: direct instillationduring surgery; direct injection into the injured tissue or organ;indirect delivery through a catheter to the injured tissue or organ. Thecomposition can also be delivered as a liquid, gel or in a solidformulation, such as a graft or patch or associated with a cellularscaffold or a particulate. Dosages and frequency will vary dependingupon the needs of the patient and judgment of the physician.

In certain embodiments, the present invention provides a native skeletalmuscle ECM decellularization and gelation method to create an in situscaffold for cellular transplantation. An appropriate digestion andpreparation protocol has been provided herein that can createnanofibrous gels. The gel solution is capable of being parenterallydelivered into the skeletal muscle tissue or other injured tissue ororgan, thus providing an in situ gelling scaffold. Since adecellularized skeletal muscle ECM best mimics the natural skeletalmuscle environment, it improves cell survival and retention uponinjection at the site of the injured tissue, thus encouraging tissueregeneration.

The skeletal muscle ECM of the present invention can also be used torecruit cells into the injured tissue or as a drug delivery vehicle. Insome instances, the composition herein can recruit endogenous cellswithin the recipient and can coordinate the function of the newlyrecruited or added cells, allowing for cell proliferation or migrationwithin the composition. An extracellular matrix composition herein canfurther comprise one or more additional components, for example withoutlimitation: an exogenous cell, a peptide, polypeptide, or protein, avector expressing a DNA of a bioactive molecule, and other therapeuticagents such as drugs, cellular growth factors, nutrients, antibiotics orother bioactive molecules. Therefore, in certain preferred embodiments,the ECM composition can further comprise an exogenous population ofcells such as stem cells or progenitor, or skeletal muscle cellprecursors, as described below.

The skeletal muscle ECM of the present invention can be derived from thenative or natural matrix of mammalian skeletal muscle tissue. Theskeletal muscle ECM of the present invention can also be derived from ananimal or synthetic source. In some instances, the decellularizedskeletal muscle extracellular matrix is derived from native skeletalmuscle tissue selected from the group consisting of human, porcine,bovine, goat, mouse, rat, rabbit, or any other mammalian or animalskeletal muscle. In some embodiments, the biocompatible compositioncomprising the decellularized skeletal muscle extracellular matrix is inan injectable gel or solution form, and can be used for skeletal muscleor other tissue repair by transplanting or delivering cells containedtherein into the injured or desired tissue in need following a diseasecondition, or recruiting the patient's own cells into the injured ordesired tissue in need. In other instances, the biocompatible materialcomprising a decellularized skeletal ECM is, for example, a patch, anemulsion, a viscous liquid, fragments, particles, microbeads, ornanobeads.

In some instances, the invention provides biocompatible materials forculturing skeletal muscle cells or other skeletal muscle relevant cellsin research laboratories, or in a clinical setting prior totransplantation and for skeletal muscle or other tissue repair. Methodsfor manufacturing and coating a surface, such as tissue culture platesor wells, with decellularized skeletal extracellular matrix are alsoprovided. The biocompatible materials of the invention are also suitablefor implantation into a patient, whether human or animal.

The invention further provides a method of producing a biocompatiblematerial comprising the decellularized skeletal muscle extracellularmatrix of the invention. Such method comprises the steps of: (a)obtaining a skeletal muscle tissue sample having an extracellular matrixcomponent and non-extracellular matrix component; (b) processing theskeletal muscle tissue sample to remove at least a portion orsubstantially all the non-extracellular matrix component to obtaindecellularized skeletal muscle extracellular matrix; and (c) sterilizingthe decellularized skeletal muscle extracellular matrix. In certainembodiments, the skeletal muscle tissue sample is isolated from a mammalsuch as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.)or a primate (e.g., monkey and human), or an avian source (e.g.,chicken, duck, etc.). Decellularization procedures for the skeletaltissue sample are performed using one or more physical, chemical and/orbiological techniques, known in the art and as taught herein. Methods ofmaking the compositions herein can include decellularizing tissue fromany age animal or human by methods well known in the art.

For human therapy, there are many potential sources for the skeletalmuscle extracellular matrix material: human skeletal muscle (includingautologous, allogeneic, or cadaveric), porcine skeletal muscle, bovineskeletal muscle, goat skeletal muscle, mouse skeletal muscle, ratskeletal muscle, rabbit skeletal muscle, chicken skeletal muscle, andother animal sources. One donor skeletal muscle can be used to treatmany people. Non-human animals are a source of skeletal muscleextracellular matrix without the need for human donors. As a researchreagent, non-human animal sources can be utilized.

In certain embodiments, the method of processing the skeletal muscleextracellular matrix is as follows. The skeletal tissue is firstdecellularized, leaving only the extracellular matrix. Decellularizationcan be performed with a perfusion of sodium dodecyl sulfate andphosphate buffered solution, or other detergents, for example. Theskeletal muscle extracellular matrix is then lyophilized, ground up, anddigested with pepsin at a low pH, between about pH 1-6 or pH 1-4, orother matrix degrading enzymes such as matrix metalloproteinases.

To produce a gel form of the skeletal muscle extracellular matrix for invivo therapy, the solution comprising the skeletal muscle extracellularmatrix is then neutralized and brought up to the desired temperature,concentration and viscosity using PBS/saline. In certain embodiments,the ECM concentration can be 1-20 mg/mL, or 2-8 mg/mL. The solutioncomprising the skeletal muscle extracellular matrix can then be injectedthrough a high gauge needle, such as 27 gauge or higher, into theinjured tissue or any tissue in need. At body temperature, e.g., 36.8°C.±0.7° C., such solution then forms into a gel. Cells, drugs, proteins,or other therapeutic agents can also be delivered inside the skeletalmuscle ECM gel.

To produce a gel form of the skeletal muscle extracellular matrix for invitro uses, the solution comprising the skeletal muscle extracellularmatrix is neutralized and brought up to the desired concentration usingPBS/saline. In certain embodiments, the ECM concentration can be 1-20mg/mL, or 2-8 mg/mL. Such solution can then be placed onto any solidsurface such as into tissue culture plates/wells. Once placed in anincubator at 37° C. or above room temperature, the solution forms a gelthat can be used for cell culture.

The invention also provides a therapeutic method for skeletal muscle orother relevant tissue repair in a subject comprising injecting orimplanting in part or in its entirety the biocompatible skeletal muscleECM material of the invention into a patient. The invention furtherprovides a therapeutic method for preventing or treating degenerativemuscles or other defective, diseased, damaged, or injured tissue ororgan in a subject comprising injecting or implanting the biocompatiblematerial of the invention in situ.

The compositions herein can comprise a decellularized ECM derived fromskeletal muscle tissue and another component or components. In someinstances, the amount of ECM in the total composition is greater than90% or 95% or 99% of the composition by weight. In some embodiments, theECM in the total composition is greater than 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, or 80% of the composition by weight. Decellularizedextracellular matrices are prepared such that much of the bioactivityfor skeletal muscle tissue regeneration is preserved. Exemplarybioactivity of the compositions herein include without limitation:control or initiation of cell adhesion, cell migration, celldifferentiation, cell maturation, cell organization, cell proliferation,cell death (apoptosis), stimulation of angiogenesis, proteolyticactivity, enzymatic activity, cell motility, protein and cellmodulation, activation of transcriptional events, provision fortranslation events, inhibition of some bioactivities, for exampleinhibition of coagulation, stem cell attraction, chemotaxis, and MMP orother enzyme activity.

The compositions comprise an extracellular matrix that is substantiallydecellularized. In some instances, a decellularized matrix comprises noliving native cells with which the ECM naturally occurs. In someinstances, a substantially decellularized matrix comprises less than 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% native cells by weight.

As described herein, a composition can comprise a decellularizedskeletal muscle ECM and different tissue decellularized EMC or asynthetic or naturally occurring polymer from animal and non-animalsources (such as plants or synthetic collagens). For example, acomposition herein comprises a natural polymer such as collagen,chitosan, alginate, glycosaminoglycans, fibrin, or hyaluronic acid. Inanother example, a composition herein comprises a synthetic polymer, forexample without limitation, polyethylene glycol, poly(glycolic)acid,poly(lactic acid), poly(hydroxy acids), polydioxanone, polycaprolactone,poly(ortho esters), poly(anhydrides), polyphosphazenes, poly(aminoacids), pseudo-poly(amino acids), conductive polymers (such aspolyacetylene, polypyrrole, polyaniline), or polyurethane or theirpotential copolymers. In some instances, a composition here comprise ECMand both a natural and a synthetic polymer. A composition herein can bea multi-material by linking an ECM and another polymer material, forexample, via reaction with amines, free thiols, or short peptides thatcan be self-assembled with the ECM.

In some instances, a polymer of the composition is biocompatible andbiodegradable and/or bioabsorbable, and can be a random copolymer, blockcopolymer or blend of monomers, homopolymers, copolymers, and/orheteropolymers that contain these monomers. Exemplary biodegradable orbioabsorbable polymers include, but are not limited to: polylactides,poly-glycolides, polycarprolactone, polydioxane and their random andblock copolymers. A biodegradable and/or bioabsorbable polymer cancontain a monomer selected from the group consisting of a glycolide,lactide, dioxanone, caprolactone, trimethylene carbonate, ethyleneglycol and lysine. The biodegradable and/or bioabsorbable polymers cancontain bioabsorbable and biodegradable linear aliphatic polyesters suchas polyglycolide (PGA) and its random copolymerpoly(glycolide-co-lactide-) (PGA-co-PLA). Other examples of suitablebiocompatible polymers are polyhydroxyalkyl methacrylates includingethylmethacrylate, and hydrogels such as polyvinylpyrrolidone andpolyacrylamides. Other suitable bioabsorbable materials are biopolymerswhich include collagen, gelatin, alginic acid, chitin, chitosan, fibrin,hyaluronic acid, dextran, polyamino acids, polylysine and copolymers ofthese materials. Any combination, copolymer, polymer or blend thereof ofthe above examples is contemplated for use according to the presentinvention. Such bioabsorbable materials may be prepared by knownmethods.

Therefore, methods are described herein for preparing a compositioncomprising decellularized ECM derived from skeletal muscle tissue. Theinvention also provides ECM compositions and methods derived fromskeletal muscle tissue in an analogous process. Related compositions,devices and methods of production and use also are provided. In someinstances a composition comprises crosslinkers including, but notlimited to, common collagen crosslinkers, hyaluronic acid crosslinkers,or other protein cross-linkers with altered degradation and mechanicalproperties. The compositions which may include cells or othertherapeutic agents may be implanted into a patient, human or animal, bya number of methods. In some instances, the compositions are injected asa liquid into a desired site in the patient.

In certain embodiments, the viscosity of the composition increases whenwarmed above room temperature including physiological temperaturesapproaching about 37° C. According to one non-limiting embodiment, theECM-derived composition is an injectable solution at room temperatureand other temperatures below 35° C. In another non-limiting embodimentthe gel can be injected body temperature above about 37° C. or near bodytemperature, but gels more rapidly at increasing temperatures. A gelforms after approximately 15-20 minutes at physiological temperature of37° C. A general set of principles for preparing an ECM-derived gel isprovided along with preferred specific protocols for preparing gels inthe following Examples which are applicable and adaptable to numeroustissues including without limitation the skeletal muscle.

Commercially available ECM preparations can also be combined in themethods, devices and compositions described herein. In one embodiment,the ECM is derived from small intestinal submucosa (SIS). Commerciallyavailable preparations include, but are not limited to, SURGISIS™,SURGISIS-ES™, STRATASIS™, and STRATASIS-ES™ (Cook Urological Inc.;Indianapolis, Ind.) and GRAFTPATCH™ (Organogenesis Inc.; Canton, Mass.).In another embodiment, the ECM is derived from dermis. Commerciallyavailable preparations include, but are not limited to PELVICOL™ (soldas PERMACOL™ in Europe; Bard, Covington, Ga.), REPLIFORM™ (Microvasive;Boston, Mass.) and ALLODERM™ (LifeCell; Branchburg, N.J.).

In some instances, the solution, gel form, and adsorbed form of theskeletal muscle extracellular matrix of the invention provide all theconstituents at the similar ratios found in vivo. For therapeutictreatment, the skeletal muscle extracellular matrix of the invention canbe delivered which can allow for skeletal muscle or other relevanttissue repair or regeneration. For in vitro cell culture for skeletalmuscle cells and other relevant cells, the gel and adsorbed forms of theskeletal muscle extracellular matrix of the invention contain all ormany of the same extracellular matrix cues that the cells recognize invivo as compared to the commonly used collagen, laminin, SURECOAT(CELLUTRON, mixture of collagen and laminin), and gelatin.

The compositions herein provide a particulate, powder, emulsion, gel orsolution form of skeletal muscle extracellular matrix, and the use ofthese forms of skeletal muscle extracellular matrix for skeletal muscleor other relevant tissue repair or regeneration, for prevention ortreatment of certain diseases. In one embodiment, the skeletal muscletissue is first decellularized, leaving only the extracellular matrix.The matrix is then lyophilized, ground or pulverized into a fine powder,and solubilized with pepsin or other enzymes, such as, but not limitedto, matrix metalloproteases, collagenases, and trypsin.

For gel therapy, the solution is then neutralized and brought up to theappropriate concentration using PBS/saline. In one embodiment, thesolution can then be injected through a needle into the injured tissueor a tissue in need. The needle size can be without limitation 22 g, 23g, 24 g, 25 g, 26 g, 27 g, 28 g, 29 g, 30 g, or smaller. In oneembodiment, the needle size through which the solution is injected is 27g. Delivery can also occur through a balloon infusion catheter or othernon-needle catheter. Dosage amounts and frequency can routinely bedetermined based on the varying condition of the injured tissue andpatient profile. At body temperature, the solution can then form into agel. In yet another embodiment, gel can be crosslinked withglutaraldehye, formaldehyde, bis-NHS molecules, or other crosslinkers.

In yet another embodiment, the ECM can be combined with othertherapeutic agents, such as cells, peptides, proteins, DNA, drugs,nutrients, antibiotics, survival promoting additives, proteoglycans,and/or glycosaminolycans. In yet another embodiment, the ECM can becombined and/or crosslinked with a synthetic polymer. Examples ofsynthetic polymers include, but are not limited to: polyethyleneterephthalate fiber (DACRON™), polytetrafluoroethylene (PTFE),polylactic acid (PLA), polyglycolic acid (PGA), polyethylene glycol(PEG), poly(ethylene glycol) diacrylate (PEG diacrylate), polyethylene,polystyrene and nitinol.

In yet another embodiment, ECM solution or gel can be injected into theinjured tissue or other relevant tissue in need, alone or in combinationwith above-described components for endogenous cell ingrowth,angiogenesis, and regeneration. In yet another embodiment, the ECM orECM liquid can be sprayed on or into injured tissue or other relevanttissue in need, alone or in combination with above-described componentsfor endogenous cell ingrowth, angiogenesis, and regeneration. In yetanother embodiment, the composition can also be used alone or incombination with above-described components as a matrix to changemechanical properties of the skeletal muscle or other relevant tissuesand/or to restore muscle mass and function. In yet another embodiment,the composition can be delivered with cells alone or in combination withthe above-described components for regenerating muscle mass andfunction. In yet another embodiment, the composition can be used aloneor in combination with above-described components for increasingarteriole and capillary density, as well as recruiting more desiredcells for tissue repair and regeneration.

In one embodiment for making a soluble reagent, the solution is broughtup in a low pH, neutral pH, or physiological pH solution including butnot limited to 0.5 M, 0.1, or 0.01 M acetic acid or 0.1M HCl, PBS, orother buffering solutions to the desired concentration and then placedinto tissue culture plates/wells, coverslips, scaffolding or othersurfaces for tissue culture. After placing in an incubator at 37° C. for1 hour, or overnight at room temperature, the excess solution isremoved. After the surfaces are rinsed with PBS, cells can be culturedon the adsorbed matrix. The solution can be combined in advance withpeptides, proteins, DNA, drugs, nutrients, survival promoting additives,proteoglycans, and/or glycosaminoglycans before, during, or afterinjection/implantation.

The present invention provides enhanced cell attachment and survival onboth the therapeutic composition and adsorbed cell culturing compositionforms of the skeletal muscle extracellular matrix in vitro. The solublecell culturing reagent form of the skeletal muscle extracellular matrixinduces faster spreading, faster maturation, and/or improved survivalfor skeletal muscle cells and other relevant cells compared to standardplate coatings.

In an embodiment herein, a biomimetic ECM derived from native skeletalmuscle tissue is disclosed. In some instances, a matrix resembles the invivo skeletal muscle or other relevant tissue environment in that itcontains many or all of the native chemical cues found in naturalskeletal muscle ECM. In some instances, through crosslinking or additionor other materials, the mechanical properties of healthy adult orembryonic skeletal muscle can also be mimicked. As described herein,skeletal muscle ECM can be isolated and processed into a gel using asimple and economical process, which is amenable to scale-up forclinical translation.

In some instances, a composition as provided herein can comprise amatrix and exogenously added or recruited cells. The cells can be anyvariety of cells. In some instances, the cells are a variety of skeletalmuscle or relevant cells including, but not limited to: stem cells,progenitors, skeletal muscle precursor cells, and fibroblasts derivedfrom autologous or allogeneic sources.

The invention thus provides a use of a gel made from nativedecellularized skeletal muscle extracellular matrix to support isolatedneonatal skeletal muscle or stem cell progenitor derived skeletal musclecells in vitro and to act as an in situ gelling scaffold, providing anatural matrix to improve cell retention and muscle mass restoration. Ascaffold created from skeletal muscle ECM is well-suited for celltransplantation in the injured tissue, since it more closelyapproximates the in vivo environment compared to currently availablematerials.

A composition herein comprising skeletal muscle ECM and exogenouslyadded cells can be prepared by culturing the cells in the ECM. Inaddition, where proteins such as growth factors are added into theextracellular matrix, the proteins may be added into the composition, orthe protein molecules may be covalently or non-covalently linked to amolecule in the matrix. The covalent linking of protein to matrixmolecules can be accomplished by standard covalent protein linkingprocedures known in the art. The protein may be covalently or linked toone or more matrix molecules.

In one embodiment, when delivering a composition that comprises thedecellularized skeletal muscle ECM and exogenous cells, the cells can befrom cell sources for treating certain diseases that include allogeneic,xenogeneic, or autogenic sources. Accordingly, embryonic stem cells,fetal or adult derived stem cells, induced pluripotent stem cells,skeletal muscle progenitors, fetal and neonatal skeletal muscle cells,mesenchymal cells, parenchymal cells, epithelial cells, endothelialcells, mesothelial cells, fibroblasts, hematopoietic stem cells, bonemarrow-derived progenitor cells, skeletal cells, macrophages,adipocytes, and autotransplanted expanded skeletal cells can bedelivered by a composition herein. In some instances, cells herein canbe cultured ex vivo and in the culture dish environment differentiateeither directly to skeletal muscle cells, or to bone marrow cells thatcan become skeletal muscle cells. The cultured cells are thentransplanted into the mammal, either with the composition or in contactwith the scaffold and other components.

Adult stem cells are yet another species of cell that can be part of acomposition herein. Adult stem cells are thought to work by generatingother stem cells (for example those appropriate to skeletal muscle) in anew site, or they differentiate directly to a skeletal muscle cells invivo. They may also differentiate into other lineages after introductionto organs, such as the skeletal muscle. The adult mammal providessources for adult stem cells in circulating endothelial precursor cells,bone marrow-derived cells, adipose tissue, or cells from a specificorgan. It is known that mononuclear cells isolated from bone marrowaspirate differentiate into endothelial cells in vitro and are detectedin newly formed blood vessels after intramuscular injection. Thus, useof cells from bone marrow aspirate can yield endothelial cells in vivoas a component of the composition. Other cells which can be employedwith the invention are the mesenchymal stem cells administered withactivating cytokines. Subpopulations of mesenchymal cells have beenshown to differentiate toward skeletal muscle generating cell lines whenexposed to cytokines in vitro.

Human embryonic stem cell derived skeletal muscle cells can be grown ona composition herein comprising the skeletal muscle ECM of the presentinvention. In some instances, hESC-derived skeletal muscle cells grownin the presence of a composition herein provide a more in vivo-likemorphology. In some instances, hESC-derived skeletal muscle cells grownin the presence of a composition herein provide increased markers ofmaturation.

The invention is also directed to a drug delivery system comprisingdecellularized skeletal muscle extracellular matrix for deliveringcells, drugs, molecules, or proteins into a subject for treatingdefective, diseased, damaged, ischemic, ulcer or other injured tissuesor organs. In one embodiment, the inventive biocompatible skeletalmuscle ECM material comprising the decellularized skeletal muscleextracellular matrix alone or in combination with other components isused for treating muscle degeneration, and other diseases to increasearteriole and capillary density and restore muscle mass and function.Therefore, the inventive biocompatible ECM material can be used totransplant cells, or injected alone to recruit native cells or othercytokines endogenous therapeutic agents, or act as a exogenoustherapeutic agent delivery vehicle.

The composition of the invention can further comprise cells, drugs,proteins, or other biological material such as, but not limited to,erythropoietin (EPO), stem cell factor (SCF), vascular endothelialgrowth factor (VEGF), transforming growth factor (TGF), fibroblastgrowth factor (FGF), epidermal growth factor (EGF), cartilage growthfactor (CGF), nerve growth factor (NGF), keratinocyte growth factor(KGF), skeletal growth factor (SGF), osteoblast-derived growth factor(BDGF), hepatocyte growth factor (HGF), insulin-like growth factor(IGF), cytokine growth factor (CGF), stem cell factor (SCF),platelet-derived growth factor (PDGF), endothelial cell growthsupplement (EGGS), colony stimulating factor (CSF), growthdifferentiation factor (GDF), integrin modulating factor (IMF),calmodulin (CaM), thymidinc kinase (TK), tumor necrosis factor (TNF),growth hormone (GH), bone morphogenic proteins (BMP), matrixmetalloproteinase (MMP), tissue inhibitor matrix metalloproteinase(TIMP), interferon, interleukins, cytokines, integrin, collagen,elastin, fibrillins, fibronectin, laminin, glycosaminoglycans,hemonectin, thrombospondin, heparan sulfate, dermantan, chondroitinsulfate (CS), hyaluronic acid (HA), vitronectin, proteoglycans,transferrin, cytotactin, tenascin, and lymphokines.

Tissue culture plates can be coated with either a soluble ligand or gelform of the extracellular matrix of the invention, or an adsorbed formof the extracellular matrix of the invention, to culture skeletal musclecells or other cell types relevant to skeletal muscle tissue or otherrelevant tissue repair. This can be used as a research reagent forgrowing these cells or as a clinical reagent for culturing the cellsprior to implantation. The extracellular matrix reagent can be combinedwith other tissue matrices and cells.

For gel reagent compositions, the solution is then neutralized andbrought up to the appropriate concentration using PBS/saline or otherbuffer, and then be placed into tissue culture plates and/or wells. Onceplaced in an incubator at 37° C., the solution forms a gel that can beused for any 2D or 3D culture substrate for cell culture. In oneembodiment, the gel composition can be crosslinked with glutaraldehye,formaldehyde, bis-NHS molecules, or other crosslinkers, or be combinedwith cells, peptides, proteins, DNA, drugs, nutrients, survivalpromoting additives, proteoglycans, and/or glycosaminolycans, orcombined and/or crosslinked with a synthetic polymer for further use.

The invention further provides an exemplary method of culturing cellsadsorbed on a decellularized skeletal muscle extracellular matrixcomprising the steps of: (a) providing a solution comprising thebiocompatible material of decellularized skeletal muscle ECM in low pHsolution or approximately neutral or physiological pH including but notlimited to, 0.5 M, or 0.01 M acetic acid or 0.1M HCl or PBS or any otherbuffered solution to a desired concentration, (b) placing said solutioninto tissue culture plates or wells, (c) incubating said tissue cultureplates or wells above room temperature such as at 37° C., for between 1hour and overnight (or at room temperature to 40° C.), (d) removingexcess solution, (e) rinsing said tissue culture plates or wells withPBS, and (f) culturing cells on the adsorbed matrix. Cells that can becultured on the adsorbed matrix comprising the skeletal muscleextracellular matrix of the invention include skeletal muscle cells orother cell types relevant to skeletal muscle repair, including stemcells and skeletal muscle cell progenitors.

Skeletal myoblasts plated on skeletal muscle matrix displayed asignificant increase in i) the number of myosin heavy chain positivemyotubes, ii) the number of nuclei per myotube and iii) myotube widthwhen compared to cells plated on traditional collagen type I coatedsubstrates. In some instances, the compositions are configured toprovide the ability to reconstitute the in vivo muscle ECM. Thecomposition may provide a tool to assess and maintain muscle and stemcell behavior in vitro similar to the native state, and may provide atool for cell-mediated therapies in vivo.

In one instance, a method of making the composition herein compriseselectrospinning. In some instances, a method herein is configured tocontrol the nanofiber size, shape, or thickness. In some instances,contractility can be induced into the composition, for example, withcells or external pacing. Contractility can create cyclic stress topromote a more natural skeletal muscle. In some instances, cell influxand angiogenesis can be induced into the composition, for example, whenthe composition comprises linked groups or embedded factors, such asangiogenic factors.

In some instances, a composition herein may contain microbeads.Microbeads can be a part of the composition or delivered by thecomposition. Exemplary microbeads can be any variety of materials, forexample, natural or synthetic. In some instances, the microbeads canhave varied degradation properties or comprise, for example, MMPinhibitors, growth factors, or small molecules.

In some instances, the composition can comprise a biological group thatcan act as an adhesive or anchor where the composition is delivered. Inone instance, a composition can be a bioadhesive, for example, for woundrepair. In some instances, a composition herein can be configured as acell adherent. For example, the composition herein can be coating ormixed with on a medical device or a biologic that does or does notcomprises cells. For example, the composition herein can be a coatingfor a synthetic polymer graft. In some instances, the compositionincludes an anti-bacterial or anti-bacterial agents could be included.Methods herein can comprise delivering the composition as a wound repairdevice. In one instance, a composition comprises an alginate bead thatis coated with an ECM composition as described herein.

In some instances, the composition is injectable. An injectablecomposition can be, without limitation, a powder, liquid, particles,fragments, gel, or emulsion. The injectable composition can be injectedinto an injured tissue or organ. The compositions herein can recruit,for example without limitation, endothelial, smooth muscle, skeletalmuscle, progenitors, and stem cells.

The composition of the present invention can be developed for substratecoating for a variety of applications. In some instances, the ECM of thecomposition retains a complex mixture of muscle-specific ECM componentsafter solubilization. In some instances, the coatings herein can moreappropriately emulate the native muscle ECM in vitro.

In some instances, a composition herein is a coating. The coating cancomprise an ECM from any tissue for example cardiac muscle, skeletalmuscle, pericardium, liver, adipose tissue, and brain. A coating can beused for tissue culture applications, both research and clinical. Thecoating can be used to coat, for example without limitation, syntheticor other biologic scaffolds/materials, or implants. In some instances, acoating is texturized or patterned. In some instances, a method ofmaking a coating includes adsorption or chemical linking. A thin gel oradsorbed coating can be formed using an ECM solution form of thecomposition. In some instances, a composition herein is configured toseal holes in the heart such as septal defects. The compositions of thepresent invention may be used as coating for biologics, medical devicesor drug delivery devices.

The native ECM is a complex combination of fibrous proteins andproteoglycans that can affect many aspects of cellular behavior. Toregenerate tissue, a scaffold should mimic this native microenvironment.The present invention, therefore, provides an injectable hydrogelderived from skeletal muscle ECM, which mimics the native biochemicalcues, as well as being amenable to minimally invasive, injectableprocedures, providing an advantage for treating muscle degeneration. Incertain embodiments, the invention can be used as a delivery vehiclecombined with cells and/or growth factors. In certain embodiments of theinvention, the compositions and methods herein provide skeletal muscleECM material as an acellular stand-alone therapy, which is used torecruit endogenous cells for neovascularization and repair. In certainembodiments, the present invention provides a porcine source of skeletalmuscle matrix. Xenogeneic decellularized extracellular matrices arebiocompatible upon removal of the cellular antigens, and can be utilizedin the clinic for a number of surgical repair applications.

A liquid version of skeletal muscle matrix herein can form a porousscaffold upon injection, which promotes cellular infiltration to thedamaged area. In certain embodiments, remnant growth factors arepresent. In other embodiments, remnant growth factors are not present.In methods herein, the decellularization and subsequent processing intothe hydrogel form decreases the probability of the presence of remnantgrowth factors.

In the present invention, mitogenic properties of the degradationproducts of the skeletal muscle ECM material were assessed on smoothmuscle cells, a relevant cell type for vascularization. The skeletalmuscle matrix degradation fragments induced a higher proliferation ratecompared to collagen. Extracellular matrix degradation productssometimes have mitogenic activity. The examples herein provide evidencethat the injectable skeletal muscle matrix scaffold inducesneovascularization in vivo.

Therefore, the ability of this present scaffold to induceneovascularization was then assessed in a rat hindlimb ischemia modelcompared to collagen, which is the predominant component of the skeletalmuscle matrix and a commonly utilized scaffold. Not only was the vesseldensity higher in the skeletal muscle matrix, but there weresignificantly more large-diameter vessels greater than 25 μm, indicatingmaturation of the vasculature. Additionally, significance was seen asearly as three days post-injection demonstrating the fast rate ofvascularization. The presence of more mature vasculature indicatespermanence of the formed vessels, which is important to getting avascular supply as quickly as possible to the ischemic region, and tomaintain blood flow (Banker and Goslin, 1998).

In certain embodiments, the liquid form of decellularized skeletalmuscle, when utilized as a coating for cell culture, increased skeletalmyoblast differentiation compared to collagen coatings. In certainembodiments, the composition provides tissue specific biochemical cuesto recapitulate the skeletal muscle microenvironment. The inventiondemonstrated that the degradation products of this scaffold increasedmyoblast proliferation compared to collagen, which is consistent withliterature demonstrating the inhibitory effect of collagen on smoothmuscle cells and fibroblasts (Koyama et al., 1996; Rhudy and McPherson,1988).

Next, the infiltration of muscle cells into the scaffold in the hindlimbischemia model was assessed. The number of desmin- and MyoD-positivecells that were recruited into the skeletal muscle matrix scaffold wasmeasured as compared to that into the collagen. Desmin, a musclespecific protein, confirms that the cells that infiltrated were from amyogenic origin. MyoD, on the other hand, is a specific striated muscleregulatory transcription factor, which coordinates the myogenic programin differentiating myoblasts (Kanisicak et al., 2009; Lee et al., 2000;Wada et al., 2002). The invention provides that there were asignificantly higher number of muscle cell types in the skeletal musclematrix, and that many of these cells were also proliferating. TheMyoD-positive cells also indicate that immature progenitor cell typesare recruited to the skeletal muscle matrix. The presence of theseMyoD-positive and desmin-positive muscle cells indicate that theskeletal muscle scaffold is recruiting relevant cell types that aid inthe regeneration of the damaged muscle, in addition to treating theischemic tissue.

The present invention, thus, provides an acellular, biomaterial-onlytherapy for treating muscle degeneration. Previous biomaterialstrategies have only utilized scaffolds to enhance cell or growth factortherapy (Doi et al., 2007; Jay et al., 2008; Kong et al., 2008; Laymanet al., 2007; Lee et al., 2010; Ruvinov et al., 2010; Silva and Mooney,2007). To increase its therapeutic benefit, the invention can be used inconjunction with cell or growth factor therapy as these components canbe added to the biomaterial prior to injection. To create a materialthat could be easily prepared in the clinic, a method that allowed forlong-term storage of the injectable skeletal muscle matrix scaffold wasalso developed with only sterile water required to resuspend itimmediately prior to use.

The invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. It is apparent for skilled artisans that variousmodifications and changes are possible and are contemplated within thescope of the current invention.

EXAMPLES Example 1 Injectable Skeletal Muscle Matrix

Skeletal muscle matrix material was derived through decellularization ofporcine skeletal muscle tissue (FIG. 1A). Fat and connective tissue wasremoved, and the skeletal muscle was cut into ˜1 cm3 pieces, rinsed withdeioninized water and stirred in 1% (wt/vol) solution of SDS in PBS for4-5 days. The decellularized muscle was then stirred overnight indeionized water, and agitated rinses under running deionized water wereperformed to remove residual SDS. In addition to confirmation with lackof nuclei on H&E stained sections (not shown), the DNA content of thematerial was measured as 26.14±1.67 ng of DNA/mg of dry weight ECM,which confirmed decellularization. The matrix was then lyophilized (FIG.1B) and milled into a fine particulate. At this stage the material canbe hydrated and utilized for in vivo injection or it can beenzymatically digested to form a liquid (FIG. 1C). At this stage, theliquid skeletal muscle matrix can be diluted and utilized as a coatingfor cell culture, or can be brought to physiological pH and temperature,which triggers assembly into a hydrogel (FIG. 1D). After raising the pHof the material to 7.4 at room temperature, the material can also bere-lyophilized (FIG. 1E) for long-term storage at −80° C. The materialcan then be resuspended at a later date using only sterile water (FIG.1F) and utilized for in vivo injection.

Example 2 Mitogenic Assay

Degradation products of decellularized ECMs have been previously shownto have mitogenic activity. It was examined whether the degradationproducts of the skeletal muscle matrix hydrogel had a mitogenic effecton cells in vitro. Proliferation of smooth muscle cells and skeletalmyoblasts following exposure to either enzymatically degraded skeletalmuscle ECM or collagen was assessed. Pepsin was also included as acontrol, as pepsin was utilized to digest the matrix material. APicogreen assay was used to determine double stranded DNA content atdays 3, 5, and 7 in culture to quantify cell proliferation. It was foundthat both smooth muscle cells (FIG. 2A) and myoblasts (FIG. 2B), whencultured in media containing degraded skeletal muscle matrix, had ahigher rate of proliferation compared to cells cultured in mediacontaining the same concentration of collagen. The increase in cellnumber was significantly greater at all time points (p<0.01). At day 3,there was a 1.85-fold increase in cell number in the skeletal musclematrix wells compared to collagen for the smooth muscle cells, and acorresponding 2.15-fold increase with the skeletal myoblasts. There wasalso a 1.3 fold increase for skeletal muscle matrix wells compared topepsin for both cell types, while the pepsin and collagen controls werenot statistically different. Thus, degradation products of the skeletalmuscle matrix were shown to promote mitogenic activity in both celltypes in vitro when compared to collagen or the pepsin control.

Example 3 Gelation In Vitro and In Vivo

A gel form of the matrix was initially made in vitro by bringing thematerial (6 mg/mL) to a physiological pH and incubating the material at37° C. After gelation, the material was tested for rheologicalproperties where it was determined that the material had a storagemodulus (G′) of 6.5±0.5 Pa. A representative trace of rheological datais shown in FIG. 3. The ability of the liquid skeletal muscle matrix toform a gel in situ were then assessed by injecting the material into ahealthy rat hindlimb. For all in vivo studies, liquid skeletal musclematrix, which had been biotinylated and re-lyophilized for storage at−80 C were utilized. Prior to injection, the material was resuspended insterile water alone. The skeletal muscle matrix was then loaded into asyringe and injected intramuscularly into a rat hindlimb (FIG. 4A). Todetermine whether the skeletal muscle matrix would assemble and form ascaffold, the injection region was excised after 20 minutes. A visiblegel, denoted by the white region in FIG. 4B, was observed within themuscle. Additional matrix injections were cryosectioned and stained tovisualize the biotinylated matrix. The liquid skeletal muscle matrixassembled into a fibrous scaffold once in vivo (FIG. 4C). In addition,the skeletal muscle matrix was also demonstrated to form a hydrogel uponin situ injection directly into rabbit supraspinatus muscle (FIGS. 10Aand 10B). To assess the microarchitecture of the skeletal muscle matrixhydrogel, the material was injected subcutaneously, and excised after 20minutes. Scanning Electron Microscopy (SEM) demonstrated that the matrixforms a porous, fibrous scaffold, both in vitro and in vivo, that iscomposed of fibers on the nano- and micro-scale (FIG. 5).

Example 4 Cellular Infiltration and Neovascularization

Upon confirmation that the material was able to assemble upon injection,the skeletal muscle matrix hydrogel were then examined in a rat hindlimbischemia model to assess its potential. One week post-hindlimb ischemia,either skeletal muscle matrix or collagen was injected intramuscularlybelow the site of femoral artery resection. At 3, 5, 7 or 14 dayspost-injection, the muscle was harvested to determine cellularinfiltration. The hydrogel was still present at all time points,although it had significantly degraded by day 14. At each time point,the amount of neovascularization, which would be critical to treat theischemic tissue, as well as the number of muscle cells and muscleprogenitors, which could aid in repair of the damaged tissue, wereassessed.

To determine whether the acellular scaffold would support new vesselformation in vivo, smooth muscle cells in collagen (FIG. 6A) andskeletal muscle matrix (FIG. 6B) injected regions were labeled viaimmunohistochemistry. Arteriole density was significantly greater in theskeletal muscle matrix injection region compared to collagen at 3, 5,and 7 days post-injection (FIG. 6C), with many of the vessels having anaverage diameter greater than 25 μm (FIG. 6D). While not significant,there was still a distinct trend towards an increase in vasculature atday 14 following injection of the skeletal muscle matrix hydrogel.Additionally, endothelial cell infiltration was measured in collagen(FIG. 7A), and skeletal muscle matrix (FIG. 7B) injection regions.Endothelial cell density was found to be similar across all four timepoints, but was significantly greater in the skeletal muscle matrixinjection region at 3 and 7 days post-injection (FIG. 7C).

It was then determined whether muscle cells were also recruited to theinjection site using staining against desmin (FIG. 8A, 8B). The desminpositive cells were also co-stained for Ki67, a marker forproliferation, as denoted by the arrows in FIGS. 8A, 8B. The skeletalmuscle matrix recruited significantly more desmin-positive cells whencompared to the collagen matrix at 3, 5, and 7 days post-injection, athe trend that continued at day 14 (FIG. 8C). Moreover, the majority ofcells expressing desmin also were Ki67 positive, indicatingproliferating muscle cells were infiltrating the injection region (FIG.8D). The number of Ki67 and desmin positive cells was significantlyincreased at 3, 5, and 7 days post-injection, with the same trend at day14. Cell infiltration was further assessed using MyoD as a marker forthe potential recruitment of activated satellite cells. There was a lownumber of MyoD positive cells recruited into the injection region ofboth materials at the examined time points; however, there was astatistically significant increase in MyoD positive cells in theskeletal muscle matrix (FIG. 9). The MyoD staining was prevalentlyperinuclear, which has been shown in other studies.

Example 5 Injectable Skeletal Muscle Matrix Powder

After milling, the skeletal muscle matrix powder is hydrated withsterile water, saline, or PBS and injected or implanted into the injuredlimb. Depending on the concentration, the injected skeletal musclematrix can form a bolus or spread throughout the tissue. Uponimplantation, the skeletal muscle ECM particulate forms a scaffold andcreates degradation products that recruit endogenous cells to repair theischemic region. These cells include blood vessels, skeletal musclecells, and skeletal muscle progenitors. By recruiting endogenous cellsfor repair and regeneration, the skeletal muscle matrix particulate hasto potential to treat peripheral artery disease and critical limbischemia, and the various complications associated with these diseases.

Example 6 Decellularization of Skeletal Muscle for Matrix Preparation

Skeletal muscle from the hindleg was harvested from Yorkshire pigs,approximately 30-45 kg, immediately after sedation with aketamine/xylazine combination (25 mg/kg, 2 mg/kg respectively) andeuthanasia with beuthanasia (1 mL/5 kg). Fat and connective tissue wasremoved, and the skeletal muscle was cut into ˜1 cm³ pieces anddecellularized. Briefly, the tissue was rinsed with deionized water andstirred in 1% (wt/vol) solution of sodium dodecyl sulfate (SDS) inphosphate buffered saline (PBS) for 4-5 days. Decellularized skeletalmuscle was stirred overnight in deionized water and then agitated rinsesunder running DI water were performed to remove residual SDS. A sampleof decellularized matrix was frozen in Tissue Tek O.C.T. freezingmedium, sectioned into 10 μm slices, and stained with hematoxylin andeosin (H&E) to confirm the absence of nuclei. Following thedecellularization protocol, the ECM was lyophilized overnight and milledto a fine powder using a Wiley Mini Mill. Additionally, to quantify DNAcontent, the DNeasy assay (Qiagen, Valencia, Calif.) was performedaccording to manufacturer's instructions. After extraction, the Take3plate was used to measure the concentration of DNA using a Synergy 2microplate reader (Biotek, Winooski, Vt.).

Preparation of Injectable Skeletal Muscle Matrix and Collagen

In order to render the decellularized extracellular matrix (ECM) into aliquid form, the milled form of the matrix was subjected to enzymaticdigestion. Pepsin (SIGMA, St. Louis, Mo.) was dissolved in 0.1 Mhydrochloric acid (HCl) to make a 1 mg/ml pepsin solution and thenfiltered through a 0.22 μm filter (Millipore, Billerca, Mass.). The ECMat a ratio of 10:1 was digested in the pepsin solution under constantstirring. After approximately 48 hours, the matrix was brought to aphysiological pH in a BSL-2 safety cabinet, and then either diluted forin vitro assays or for injection. For in vitro and in vivo studies, theskeletal muscle matrix was brought to a pH of 7.4 through the additionof sterile-filtered sodium hydroxide (NaOH) and 10×PBS, and furtherdiluted to 6 mg/ml using 1×PBS inside a BSL-2 safety cabinet.

Skeletal Muscle Matrix In Vitro Gel Characterization

Gels of the skeletal muscle matrix were formed, at a concentration 6mg/ml for rheological characteristics and for scanning electronmicroscopy. Either 100 μl of matrix was pipetted into a 96 well plate(Corning, Corning, N.Y.) or 500 μl in glass scintillation vials andincubated overnight to form gels. Rheometry was conducted on the 500 μlin vitro-formed skeletal muscle matrix gels using a TA instruments AR-G2rheometer. The gels were tested using a 20 mm parallel plate geometrywith a 1.2 mm gap at 37° C. Three frequency sweeps were performed withinthe linear viscoelastic strain region. Samples were run in triplicateand then the values were averaged to calculate the storage modulus.

Scanning electron microscopy (SEM) was utilized to determine themicrostructure of the skeletal muscle matrix gels. These gels wereeither formed in vivo by injecting the skeletal muscle matrixsubcutaneously in a rat and excised after 20 minutes, or in vitro afterincubation of the material in a 96 well plate at 37° C. overnight. Theskeletal muscle matrix gels were harvested and fixed with 2.5%glutaraldehyde for 2 hours, and then dehydrated using a series ofethanol rinses (30-100%). Samples were then critical point dried andcoated with iridium using an Emitech K575X Sputter coater. Electronmicroscopy images were taken using a Phillips XL30 Environmental SEMField Emission microscope at 10 kV, with 242 μA and a working distanceof 10 mm

In Vitro Proliferation Assays

Primary rat aortic smooth muscle cells (RASMC) and C2C12 skeletalmyoblasts were maintained on collagen coated plates and split at 1:5every 2-3 days. Cells between passages 4 and 10 were plated at 750cells/well in 96 well plates in growth media consisting of DMEM, 10%fetal bovine serum, and 1% pen-strep solution. Twenty-four hours later,the cells were washed with PBS to remove non-adherent cells. Digestedskeletal muscle matrix and collagen were brought to a pH of 7.4, andthen added to the growth media at concentrations of 0.05 mg/mL. As theECM was enzymatically digested, pepsin was also included as a control at0.005 mg/mL. All conditions were run in quadruplicate. Every two days,media was changed and cell proliferation was assessed using thePICOGREEN® assay (Invitrogen) per manufacturer's directions. Briefly,wells were rinsed in PBS and then incubated with 100 μL of TE buffer.After incubation for 30 minutes at room temperature followed by 5minutes on a shaker, 100 μL of 1:200 Picogreen reagent was added. Uponcovering the plates in foil and shaking them for 30 minutes, doublestranded DNA was quantified using a fluorescent plate reader at 630 nmat days 3, 5, and 7.

In Vivo Gelation Test

To prepare for in vivo studies, a preliminary test was performed toensure that the skeletal muscle matrix would be able to gel uponinjection. The skeletal muscle matrix was labeled with biotin, and theninjected into the hindlimbs of healthy Sprague Dawley rats. For biotinlabeling, a 10 mM solution of EZ link Sulfo-NHS-Biotin (Pierce,Rockford, Ill.) was prepared and mixed with the liquid skeletal musclematrix for a final concentration of 0.3 mg of biotin/mg matrix. Themixture was allowed to sit on ice for two hours. The skeletal musclematrix was then frozen, lyophilized and stored at −80° C. until use. Toresuspend the skeletal muscle matrix, sterile water was added at theoriginal volume to bring the material to 6 mg/ml and vortexed. FemaleHarlan Sprague Dawley rats (225-250 g) were anesthetized usingisoflurane at 5%, intubated, and maintained at 2.5% isoflurane duringsurgery. In preliminary studies, (n=2) 150 μl of skeletal muscle matrixwas injected intramuscularly into healthy rats. The muscle was excisedafter 20 min, and fresh frozen using Tissue Tek O.C.T.

Hindlimb Ischemia Model

After confirmation of gelation in vivo, a rat hindlimb ischemia modelwas utilized to test the skeletal muscle extracellular matrix. Animalswere placed in a supine position and hindlimb ischemia was induced byligation and excision of the femoral artery. After ligation of theproximal end of the femoral artery, the distal portion of the saphenousartery was ligated and the artery and side branches were dissected free,and then excised. The area was sutured closed and animals were given ananalgesic of 0.05 mg/kg of buprenorphine hydrochloride (ReckittBenckiser Healthcare (UK) Ltd., Hull, England) prior to recovery fromanesthesia. One week post-injury, the rats were anesthetized using 5%isoflurane, intubated, and maintained at 2.5% isoflurane for injection.Skeletal muscle matrix and rat tail collagen were biotinylated in orderto visualize the injection region and 150 μl was injectedintramuscularly. Injection was confirmed by a lightening of the muscleat the site of injection. Rats were sacrificed using an overdose ofsodium pentobarbital (200 mg/kg) at 3, 5, 7, or 14 days post injection(n=4, except n=3 for 14 day collagen injection), and leg muscles wereharvested and frozen in Tissue Tek O.C.T.

Histology and Immunohistochemistry

The excised muscle was cryosectioned into 10 μm slices. Slices werestained with Hematoxylin and Eosin every 1 mm and screened to determinethe location of injected material. Adjacent slides were stained forvisualization of biotin-labeled skeletal muscle matrix or collagen, toconfirm the injection site. Slides were fixed in acetone, incubated withsuperblock buffer (Pierce), followed by 3% hydrogen peroxide (Sigma),and horseradish peroxidase conjugated neutravidin (Pierce) at roomtemperature. The reaction was visualized by incubation withdiaminobenzidine (DAB, Pierce) for ten minutes.

Five slides evenly spaced within the injection region were then used forimmunohistochemistry (IHC). Sections were fixed for 2 min in acetone andblocked with staining buffer for 1 h (2% goat serum and 0.3% TritonX-100 in PBS). Skeletal muscle sections were then assessed for vesselformation using a mouse anti-smooth muscle actin antibody (Dako,Carpinteria, Calif.; 1:75 dilution) to label smooth muscle cells. Afterthree 5-minute washes with PBS, AlexaFluor 568 anti-mouse (Invitrogen,1:200 dilution) was used as a secondary. Endothelial cell infiltrationwas assessed using FITC labeled isolectin (Vector Laboratories,Burlingame, Calif.; 1:100 dilution). Slides were then mounted usingFluoromount (Sigma). Sections stained with only the primary antibody orsecondary antibody were used as negative controls. Images were taken at100× using Carl Zeiss Observer D.1 and analyzed using AxioVisionsoftware. Arterioles were quantified with a visible lumen and a diameter≥10 μm and normalized over the injection area.

In order to assess proliferating muscle cell infiltration into theinjection region, sections were stained using a mouse anti-desminantibody (Sigma; dilution 1:100) and co-stained with a rabbit anti-Ki67(Santa Cruz Biotech, Santa Cruz, Calif.; dilution 1:100). AlexaFluor 488anti-mouse and AlexaFluor 568 anti-rabbit were used for secondaryantibodies (1:200), followed by staining with Hoechst 33342. Slides weremounted with Fluoromount (Sigma) prior to imaging. Additionally, theskeletal muscle tissue was assessed using a rabbit anti-MyoD (Santa CruzBiotech, Santa Cruz, Calif.; dilution 1:100), followed by AlexaFluor 488anti-rabbit as a secondary antibody, and Hoechst 33342. Three 400×images were taken per slide and analyzed using AxioVision software. Thenumber of desmin positive cells, and desmin positive cells thatco-localized with Ki67 were counted, averaged and normalized over thearea. For the tissue sections analyzed for MyoD, the number of positivecells with MyoD co-localized with nuclei were counted and averaged overthe area of injection.

Statistical Analysis

All data is presented as the mean±standard error of mean. For the invitro assays, samples were run in quadruplicate and results wereaveraged. Significance was determined using a one-way analysis ofvariance (ANOVA) with a Bonferroni post-test. A two-tailed Student'st-test was used for all other data and reported as p<0.05 and p<0.001.

Example 7

Pelvic floor disorders (PFD) include urinary and fecal incontinence, andpelvic organ prolapse. Dysfunction of pelvic striated muscles, whichinclude external urethral (EUS) and external anal (EAS) sphinctericmuscles, and pelvic floor muscles (PFM) is a key factor in thepathogenesis of PFD. EAS and PFM dysfunction also implicated in thepathogenesis of Rectal Prolapse (RP). Furthermore, prevalence of PFD issubstantially higher on female patients with RP compared to age-matchedgeneral female population. PFM dysfunction is thought to be a commonetiological factor in the pathogenesis of these conditions.

I. Indication: Stress Urinary Incontinence (SUI) and Mixed UrinaryIncontinence (MUI).

Patient population: Patients with SUI/MUI; Patients with SUI/MUI andintrinsic sphincter deficiency; Patients with SUI/MUI and pelvic floormuscle (PFM) dysfunction; Vaginally parous women.

Time-frames: 1. at the time of primary diagnosis of bothersome SUI/MUI;2. as an adjunct therapy at the time of urethral bulking injection,sling procedure; 3. after failure to respond to other treatments for SUI(e.g. pelvic rehabilitation, urethral bulking injection, slingprocedure); 4. at the time of diagnosis of recurrent bothersome SUI; 5.at the time of vaginal delivery.

Delivery Method: a) Delivery into urethral striated sphincter;Transurethral endoscopic approach: 0, 12, or 30-degree lens can be used;Approximately 4 ml volume of material is injected containing 2-50×106MDSCs. (Smaldone et al. MINERVA UROL NEFROL 2009) the myoblasts weresuspended in 1.4 mL of Dulbecco's modified Eagle medium (DMEM)/F12 with20% autologous serum, and the fibroblasts in 1 mL DMEM/F12 with 20%autologous serum mixed with 2.5 mL of collagen (Contigen®, Bard,Covington, Ga., USA) as carrier material to prevent them from migratingfrom the site of injection, as fibroblasts are mobile after application.Using a specially designed injection device, 15-18 aliquots (50-100 μLper depot) of the myoblast suspension were injected directly into theomega-shaped rhabdosphincter at two different levels, to promoteregeneration of the muscle. Then 25-30 depots (50-100 μL per depot) ofthe fibroblast/collagen suspension were injected into the submucosacircumferentially at three levels, slightly cranial to, slightly caudalto, and between the levels of the injected myoblasts, to treat atrophyof the urethral submucosa (Mitterberger et al. BJU 2007); and b)Delivery into PFMs—levator ani muscle LAM

Indication: Anal/Fecal Incontinence (AI/FI).

Patient population: Patients with bothersome AI/FI and external analsphincter (EAS) dysfunction/atrophy (diagnosed by digital palpation,anal manometry, endoanal ultrasound); Patients with bothersome AI/FI andpelvic floor muscle (PFM) dysfunction; Vaginally parous women with thirddegree obstetrical laceration/obstetrical anal sphincter injury (OASI)

Time-frames: 1. at the time of primary diagnosis of bothersome AI/FI andEAS dysfunction; 2. as an adjunct therapy at the time of sacralneuromodulation, sphincteroplasty; 3. after failure to respond to othertreatments for AI/FI (e.g. pelvic rehabilitation, sacralneuromodulation, sphincteroplasty); 4. at the time of diagnosis ofrecurrent bothersome AI/FI; 5. at the time of vaginal deliverycomplicated by a third degree obstetrical laceration/obstetrical analsphincter injury (OASI)

Delivery Method: a) Delivery into EAS. Transcutaneous endoscopicapproach; Transrectal endoscopic approach; b) Delivery into PFMs—levatorani muscle LAM; Transvaginal approach.

Volume/dose: The amount of the ECM hydrogel provided to a patient willdepend on such factors as the amount of tissue to be treated.

Indication: Pelvic floor muscle (PFM) dysfunction.

Patient population: Patients with clinically diagnosed PFM dysfunction(clinical assessment of PFM function and by digital palpation, vaginalmanometry, pelvic imaging (levator hiatus changes with squeeze));Vaginally parous women.

Time-frames: 1. at the time of primary diagnosis of PFM dysfunction; 2.as an adjunct therapy to pelvic rehabilitation; 3. after failure torespond to other treatments for PFM dysfunction (e.g. pelvicrehabilitation); 4. at the time of diagnosis of recurrent PFMdysfunction; 5. at the time of vaginal delivery.

Delivery Method: a) Delivery into PFMs—levator ani muscle (LAM); Thelevator ani complex includes the puborectalis, pubococcygeus, andiliococcygeus muscles. Transvaginal approach (female patients):injectable hydrogel is drawn up into 1-mL syringes with 22G Yale spinalneedles attached. 5 mL of 2% lignocaine gel introduced intravaginally toprovide local anesthesia; Patients placed in lithotomy position. Themuscles to be injected are located by digital vaginal palpation. Thepuborectalis is located just distal from the hymenal ring. The needle isheld in a near horizontal plane. The vaginal epithelium is pierced justinside the hymenal ring, approximately within the posterior third of thehymenal opening. The needle is directed slightly laterally andposteriorly for approximately 5-40 mm The pubococcygeus is lateral andproximal to the puborectalis muscle. The operator places his/her indexfinger on the ischial spine and the thumb on the ishion. A spinal needleis then advanced along the outstretched index finger. The needle piercesthrough the vaginal epithelium, halfway between the ischial spine andthe hymenal ring and is advanced approximately 5-40 mm. The needle isheld pointing toward the ipsilateral gluteal region while piercingthrough the vaginal epithelium. The iliococcygeus is located proximal tothe pubococcygeus muscle. The operator places his/her index finger onthe ischial spine and the thumb on the ishion. A spinal needle is thenadvanced along the outstretched index finger. The needle pierces throughthe vaginal epithelium, ⅔ of the way proximal to the hymenal ring and isadvanced approximately 5-10 mm 1-mL aliquots injected into two sitesbilaterally within each of the puborectalis, pubococcygeus, andiliococcygeus muscles. (from the study of Botox injection for PFM spasmJarvis et al. Aust N Z J Obstet Gynaecol. 2004) PFM dysfunction can alsooccur in males, and the approach would be trasrectal

Indication: Pelvic Organ Prolapse (POP).

Patient population: Patients with bothersome POP and pelvic floor muscle(PFM) dysfunction; Vaginally parous women

Time-frames: 1. at the time of primary diagnosis of bothersome POP andpelvic floor muscle (PFM) dysfunction; 2. as an adjunct therapy at thetime of POP repair; 3. after failure to respond to other treatments forPOP (e.g. pelvic rehabilitation, pessary, surgery); 4. at the time ofdiagnosis of recurrent bothersome POP; 5. at the time of vaginaldelivery

Delivery Method: a) Delivery into PFMs—levator ani muscle LAM;Transvaginal approach.

Indication: Rectal prolapse (RP).

Patient population: Patients with bothersome RP.

Time-frames: 1. at the time of primary diagnosis of bothersome RP; 2. asan adjunct therapy at the time of RP repair; 3. after failure to respondto other treatments for RP (e.g. pelvic rehabilitation, surgery); 4. atthe time of diagnosis of recurrent bothersome RP.

Delivery Method: a) Delivery into EAS and/or PFM; Transrectal endoscopicapproach; Transvaginal approach

In addition, the skeletal muscle matrix material was demonstrated toform a hydrogel upon injection into rat pelvic floor muscles byultrasound guidance (FIGS. 11A and 11B).

The skeletal muscle matrix material was demonstrated to form a hydrogelupon injection into rat external urethral sphincter by ultrasoundguidance (FIG. 12).

Example 8

Focal skeletal muscle atrophy and degeneration: Dysfunction of striatedmuscles, which include the rotator cuff muscles (supraspinatus,infraspinatus, teres minor, subcapularis), hip abductor muscle (gluteusmedius, gluteus minimus, gluteus maximus, and short external rotators),foot and ankle muscles (tibialis posterior, gastrocnemius, soleus),lumbar spine muscles (multifidus, erector spinae), and knee extensormuscles (quadriceps), all suffer from fatty atrophy and muscledegeneration as a consequence of chronic joint disease and otherneuromuscular pathologies. This loss of muscle interferes with muscleand joint function, which negatively impacts quality of life.

Indication: Shoulder, hip, foot and ankle, lumbar spine, and kneedegenerative joint and tendon diseases, which are associated with focalmuscle atrophy and degeneration.

Patient population: Patients with Tendinopathy and/or tendon rupture,Patients with Osteoarthritis, Patients with Rheumatoid arthritis,Patients with Low Back Pain.

Time-frames: 1. at the time of primary diagnosi; 2. as an adjuncttherapy at the time of surgical repair of joint or tendon tissues; 3.after failure to respond to other treatments for muscle hypertrophy; 4.at the time of diagnosis of recurrent bothersome joint disease.

Delivery Method: Delivery into striated muscle percutaneously (needleand syringe); Delivery into striated muscle arthroscopically (needle andsyringe through an arthroscopic portal); Delivery into striated muscleduring open arthrotomy (needle and syringe through an open surgicalwound directly into muscle).

Amount of injectate: Approximately 10-50% of muscle volume.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be apparent to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

REFERENCES

-   1. Alev C, Ii M, Asahara T (2011) Endothelial progenitor cells: a    novel tool for the therapy of ischemic diseases. Antioxid Redox    Signal 15: 949-965.-   2. Bach A D, Arkudas A, Tjiawi J, Polykandriotis E, Kneser U, Horch    R E, Beier J P (2006) A new approach to tissue engineering of    vascularized skeletal muscle. J Cell Mol Med 10: 716-726.-   3. Badylak S F (2007) The extracellular matrix as a biologic    scaffold material. Biomaterials 28: 3587-3593.-   4. Badylak S F, Freytes D O, Gilbert T W (2009) Extracellular matrix    as a biological scaffold material: Structure and function. Acta    Biomater 5: 1-13.-   5. Badylak S F, Gilbert T W (2008) Immune response to biologic    scaffold materials. Semin Immunol 20: 109-116.-   6. Badylak S F, Park K, Peppas N, McCabe G, Yoder M (2001)    Marrow-derived cells populate scaffolds composed of xenogeneic    extracellular matrix. Exp Hematol 29: 1310-1318.-   7. Banker G, Goslin K (1998) Culturing Nerve Cells. The MIT Press.-   8. Beattie A J, Gilbert T W, Guyot J P, Yates A J, Badylak S    F (2008) Chemoattraction of Progenitor Cells by Remodeling    Extracellular Matrix Scaffolds. Tissue Eng Part A 15: 1119-1125.-   9. Belch J J, Topol E J, Agnelli G, Bertrand M, Califf R M, Clement    D L, Creager M A, Easton J D, Gavin J R, 3rd, Greenland P, Hankey G,    Hanrath P, Hirsch A T, Meyer J, Smith S C, Sullivan F, Weber M    A (2003) Critical issues in peripheral arterial disease detection    and management: a call to action. Arch Intern Med 163: 884-892.-   10. Bhang S H, Kim J H, Yang H S, La W G, Lee T J, Kim G H, Kim H A,    Lee M, Kim B S (2011) Combined gene therapy with hypoxia-inducible    factor-1alpha and heme oxygenase-1 for therapeutic angiogenesis.    Tissue engineering. Part A 17: 915-926.-   11. Bruey J M, Kantarjian H, Ma W, Estrov Z, Yeh C, Donahue A,    Sanders H, O'Brien S, Keating M, Albitar M (2010) Circulating Ki-67    index in plasma as a biomarker and prognostic indicator in chronic    lymphocytic leukemia. Leuk Res 34: 1320-1324.-   12. Chan Y C, Cheng S W (2011) Drug-eluting stents and balloons in    peripheral arterial disease: evidence so far. Int J Clin Pract 65:    664-668.-   13. Christman K L, Vardanian A J, Fang Q, Sievers R E, Fok H H, Lee    R J (2004) Injectable fibrin scaffold improves cell transplant    survival, reduces infarct expansion, and induces neovasculature    formation in ischemic myocardium. J Am Coll Cardiol 44: 654-660.-   14. Cooper R N, Tajbakhsh S, Mouly V, Cossu G, Buckingham M,    Butler-Browne G S (1999) In vivo satellite cell activation via Myf5    and MyoD in regenerating mouse skeletal muscle. J Cell Sci 112 (Pt    17): 2895-2901.-   15. Crapo P M, Gilbert T W, Badylak S F (2011) An overview of tissue    and whole organ decellularization processes. Biomaterials 32:    3233-3243.-   16. Dattilo P B, Casserly I P (2011) Critical limb ischemia:    endovascular strategies for limb salvage. Frog Cardiovasc Dis 54:    47-60.-   17. DeQuach J A, Mezzano V, Miglani A, Lange S, Keller G M, Sheikh    F, Christman K L (2010) Simple and high yielding method for    preparing tissue specific extracellular matrix coatings for cell    culture. PLoS One 5: e13039.-   18. Diniz G, Aktas S, Turedi A, Temir G, Ortac R, Vergin C (2011)    Telomerase reverse transcriptase catalytic subunit expression and    proliferation index in Wilms tumor. Tumour Biol 32: 761-767.-   19. Doi K, Ikeda T, Marui A, Kushibiki T, Arai Y, Hirose K, Soga Y,    Iwakura A, Ueyama K, Yamahara K, Itoh H, Nishimura K, Tabata Y,    Komeda M (2007) Enhanced angiogenesis by gelatin hydrogels    incorporating basic fibroblast growth factor in rabbit model of hind    limb ischemia. Heart Vessels 22: 104-108.-   20. Fadini G P, Agostini C, Avogaro A (2010) Autologous stem cell    therapy for peripheral arterial disease meta-analysis and systematic    review of the literature. Atherosclerosis 209: 10-17.-   21. Gilbert T W, Sellaro T L, Badylak S F (2006) Decellularization    of tissues and organs. Biomaterials 27: 3675-3683.-   22. Gupta R, Losordo D W (2011) Cell therapy for critical limb    ischemia: moving forward one step at a time. Circ Cardiovasc Intery    4: 2-5.-   23. Hidestrand M, Richards-Malcolm S, Gurley C M, Nolen G, Grimes B,    Waterstrat A, Zant G V, Peterson C A (2008) Sca-1-expressing    nonmyogenic cells contribute to fibrosis in aged skeletal muscle. J    Gerontol A Biol Sci Med Sci 63: 566-579.-   24. Jay S M, Shepherd B R, Bertram J P, Pober J S, Saltzman W    M (2008) Engineering of multifunctional gels integrating highly    efficient growth factor delivery with endothelial cell    transplantation. Faseb J 22: 2949-2956.-   25. Jeon O, Krebs M, Alsberg E (2011) Controlled and sustained gene    delivery from injectable, porous PLGA scaffolds. J Biomed Mater Res    A 98: 72-79.-   26. Kanisicak O, Mendez J J, Yamamoto S, Yamamoto M, Goldhamer D    J (2009) Progenitors of skeletal muscle satellite cells express the    muscle determination gene, MyoD. Developmental biology 332: 131-141.-   27. Kawamoto A, Katayama M, Handa N, Kinoshita M, Takano H, Horii M,    Sadamoto K, Yokoyama A, Yamanaka T, Onodera R, Kuroda A, Baba R,    Kaneko Y, Tsukie T, Kurimoto Y, Okada Y, Kihara Y, Morioka S,    Fukushima M, Asahara T (2009) Intramuscular transplantation of    G-CSF-mobilized CD34(+) cells in patients with critical limb    ischemia: a phase I/IIa, multicenter, single-blinded,    dose-escalation clinical trial. Stem Cells 27: 2857-2864.-   28. Kong H J, Kim E S, Huang Y C, Mooney D J (2008) Design of    biodegradable hydrogel for the local and sustained delivery of    angiogenic plasmid DNA. Pharm Res 25: 1230-1238.-   29. Koyama H, Raines E W, Bornfeldt K E, Roberts J M, Ross R (1996)    Fibrillar collagen inhibits arterial smooth muscle proliferation    through regulation of Cdk2 inhibitors. Cell 87: 1069-1078.-   30. Kuraitis D, Zhang P, Zhang Y, Padavan D T, McEwan K, Sofrenovic    T, McKee D, Zhang J, Griffith M, Cao X, Musaro A, Ruel M, Suuronen E    J (2011) A stromal cell-derived factor-1 releasing matrix enhances    the progenitor cell response and blood vessel growth in ischaemic    skeletal muscle. Eur Cell Mater 22: 109-123.-   31. Lawall H, Bramlage P, Amann B (2010) Stem cell and progenitor    cell therapy in peripheral artery disease. A critical appraisal.    Thromb Haemost 103: 696-709.-   32. Layman H, Rahnemai-Azar A A, Pham S M, Tsechpenakis G,    Andreopoulos F M (2011) Synergistic angiogenic effect of    codelivering fibroblast growth factor 2 and granulocyte-colony    stimulating factor from fibrin scaffolds and bone marrow    transplantation in critical limb ischemia. Tissue Eng Part A 17:    243-254.-   33. Layman H, Spiga M G, Brooks T, Pham S, Webster K A, Andreopoulos    F M (2007) The effect of the controlled release of basic fibroblast    growth factor from ionic gelatin-based hydrogels on angiogenesis in    a murine critical limb ischemic model. Biomaterials 28: 2646-2654.-   34. Lee J, Bhang S H, Park H, Kim B S, Lee K Y (2010) Active blood    vessel formation in the ischemic hindlimb mouse model using a    microsphere/hydrogel combination system. Pharm Res 27: 767-774.-   35. Lee J Y, Qu-Petersen Z, Cao B, Kimura S, Jankowski R, Cummins J,    Usas A, Gates C, Robbins P, Wernig A, Huard J (2000) Clonal    isolation of muscle-derived cells capable of enhancing muscle    regeneration and bone healing. J Cell Biol 150: 1085-1100.-   36. Li F, Li W, Johnson S, Ingram D, Yoder M, Badylak S (2004)    Low-molecular-weight peptides derived from extracellular matrix as    chemoattractants for primary endothelial cells. Endothelium 11:    199-206.-   37. Lutolf M P, Hubbell J A (2005) Synthetic biomaterials as    instructive extracellular microenvironments for morphogenesis in    tissue engineering. Nat Biotechnol 23: 47-55.-   38. Manzi M, Palena L, Cester G (2011) Endovascular techniques for    limb salvage in diabetics with crural and pedal disease. J    Cardiovasc Surg (Torino) 52: 485-492.-   39. Megeney L A, Kablar B, Garrett K, Anderson J E, Rudnicki M    A (1996) MyoD is required for myogenic stem cell function in adult    skeletal muscle. Genes Dev 10: 1173-1183.-   40. Menasché P (2010) Cell therapy for peripheral arterial disease.    Curr Opin Mol Ther 12: 538-545.-   41. Merritt E K, Hammers D W, Tierney M, Suggs L J, Walters T J,    Farrar R P Functional assessment of skeletal muscle regeneration    utilizing homologous extracellular matrix as scaffolding. Tissue Eng    Part A 16: 1395-1405.-   42. Numata S, Fujisato T, Niwaya K, Ishibashi-Ueda H, Nakatani T,    Kitamura S (2004) Immunological and histological evaluation of    decellularized allograft in a pig model: comparison with    cryopreserved allograft. J Heart Valve Dis 13: 984-990.-   43. Ott H C, Matthiesen T S, Goh S K, Black L D, Kren S M, Netoff T    I, Taylor D A (2008) Perfusion-decellularized matrix: using nature's    platform to engineer a bioartificial heart. Nat Med 14: 213-221.-   44. Reing J E, Zhang L, Myers-Irvin J, Cordero K E, Freytes D O,    Heber-Katz E, Bedelbaeva K, McIntosh D, Dewilde A, Braunhut S J,    Badylak S F (2009) Degradation products of extracellular matrix    affect cell migration and proliferation. Tissue Eng Part A 15:    605-614.-   45. Rhudy R W, McPherson J M (1988) Influence of the extracellular    matrix on the proliferative response of human skin fibroblasts to    serum and purified platelet-derived growth factor. J Cell Physiol    137: 185-191.-   46. Rieder E, Nigisch A, Dekan B, Kasimir M T, Muhlbacher F, Wolner    E, Simon P, Weigel G (2006) Granulocyte-based immune response    against decellularized or glutaraldehyde cross-linked vascular    tissue. Biomaterials 27: 5634-5642.-   47. Ruvinov E, Leor J, Cohen S (2010) The effects of controlled HGF    delivery from an affinity-binding alginate biomaterial on    angiogenesis and blood perfusion in a hindlimb ischemia model.    Biomaterials 31: 4573-4582.-   48. Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known    and the unknown. J Cell Physiol 182: 311-322.-   49. Seif-Naraghi S B, Salvatore M A, Schup-Magoffin P J, Hu D P,    Christman K L (2010) Design and characterization of an injectable    pericardial matrix gel: a potentially autologous scaffold for    cardiac tissue engineering. Tissue Eng Part A 16: 2017-2027.-   50. Silva E A, Mooney D J (2007) Spatiotemporal control of vascular    endothelial growth factor delivery from injectable hydrogels    enhances angiogenesis. J Thromb Haemost 5: 590-598.-   51. Singelyn J M, DeQuach J A, Seif-Naraghi S B, Littlefield R B,    Schup-Magoffin P J, Christman K L (2009) Naturally derived    myocardial matrix as an injectable scaffold for cardiac tissue    engineering. Biomaterials 30: 5409-5416.-   52. Singelyn J M, Sundaramurthy P, Johnson T D, Schup-Magoffin P J,    Hu D P, Faulk D M, Wang J, Mayle K M, Bartels K, Salvatore M, Kinsey    A M, Demaria A N, Dib N, Christman K L (2012) Catheter-deliverable    hydrogel derived from decellularized ventricular extracellular    matrix increases endogenous cardiomyocytes and preserves cardiac    function post-myocardial infarction. Journal of the American College    of Cardiology 59: 751-763.-   53. Sprengers R W, Lips D J, Moll F L, Verhaar M C (2008) Progenitor    cell therapy in patients with critical limb ischemia without    surgical options. Ann Surg 247: 411-420.-   54. Stansby G, Williams R (2011) Angioplasty for treatment of    isolated below-the-knee arterial stenosis in patients with critical    limb ischemia. Angiology 62: 357-358.-   55. Sundararaghavan H G, Metter R B, Burdick J A (2010) Electrospun    fibrous scaffolds with multiscale and photopatterned porosity.    Macromol Biosci 10: 265-270.-   56. Tongers J, Roncalli J G, Losordo D W (2008) Therapeutic    angiogenesis for critical limb ischemia: microvascular therapies    coming of age. Circulation 118: 9-16.-   57. Uriel S, Labay E, Francis-Sedlak M, Moya M L, Weichselbaum R R,    Ervin N, Cankova Z, Brey E M (2009) Extraction and assembly of    tissue-derived gels for cell culture and tissue engineering. Tissue    Eng Part C Methods 15: 309-321.-   58. Uygun B E, Soto-Gutierrez A, Yagi H, Izamis M L, Guzzardi M A,    Shulman C, Milwid J, Kobayashi N, Tilles A, Berthiaume F, Hertl M,    Nahmias Y, Yarmush M L, Uygun K (2010) Organ reengineering through    development of a transplantable recellularized liver graft using    decellularized liver matrix. Nat Med 16: 814-820.-   59. Valentin J E, Turner N J, Gilbert T W, Badylak S F (2010)    Functional skeletal muscle formation with a biologic scaffold.    Biomaterials 31: 7475-7484.-   60. Wada M R, Inagawa-Ogashiwa M, Shimizu S, Yasumoto S, Hashimoto    N (2002) Generation of different fates from multipotent muscle stem    cells. Development 129: 2987-2995.-   61. Webber M J, Tongers J, Newcomb C J, Marquardt K T, Bauersachs J,    Losordo D W, Stupp S I (2011) Supramolecular nanostructures that    mimic VEGF as a strategy for ischemic tissue repair. Proceedings of    the National Academy of Sciences of the United States of America    108: 13438-13443.-   62. Wolf M T, Daly K A, Reing J E, Badylak S F (2012) Biologic    scaffold composed of skeletal muscle extracellular matrix.    Biomaterials 33: 2916-2925.-   63. Yamamoto D L, Csikasz R I, Li Y, Sharma G, Hjort K, Karlsson R,    Bengtsson T (2008) Myotube formation on micro-patterned glass:    intracellular organization and protein distribution in C2C12    skeletal muscle cells. J Histochem Cytochem 56: 881-892.-   64. Young D A, Ibrahim D O, Hu D, Christman K L (2011) Injectable    hydrogel scaffold from decellularized human lipoaspirate. Acta    Biomater 7: 1040-1049.-   65. Zantop T, Gilbert T W, Yoder M C, Badylak S F (2006)    Extracellular matrix scaffolds are repopulated by bone    marrow-derived cells in a mouse model of achilles tendon    reconstruction. J Orthop Res 24: 1299-1309.

1. A method comprising injecting or implanting in a subject with muscledegeneration an effective amount of a composition comprisingdecellularized extracellular matrix derived from skeletal muscle tissue.2. The method of claim 1, wherein said composition is coated on animplant.
 3. The method of claim 1, wherein said composition is deliveredas a liquid or a powder.
 4. The method of claim 3, wherein saidcomposition transitions to a gel form after delivery.
 5. The method ofclaim 1, wherein said composition degrades within one to three monthsfollowing injection or implantation.
 6. The method of claim 1, whereininjection or implantation of said composition prevents or repairs damageto skeletal muscle tissue sustained by said subject.
 7. The method ofclaim 1, wherein injection or implantation of said composition repairsdamage caused by acute sarcomere hyperelongation and myofibrillardisruption in said subject.
 8. The method of claim 1, wherein saideffective amount is an amount that increases blood flow, increasesmuscle mass, or induces new vascular formation in the area of theinjection or implantation of the subject.
 9. The method of claim 1,wherein the effective amount is effective for treating at least one ofthe symptoms selected from the group consisting of pelvic floor muscle(PFM) fibrosis, pelvic floor disorders (PFD), urinary (UI) and fecalincontinence (H), pelvic organ prolapse (POP), dysfunction of pelvicstriated muscles, external urethral (EUS) and external anal (EAS)sphincteric muscles, rectal prolapse (RP), stress urinary incontinence(SUI), and mixed urinary incontinence (MUI).
 10. The method of claim 1,wherein the effective amount is effective for treating a birth traumaand consequent dysfunction of pelvic floor musculature.
 11. A methodcomprising injecting or implanting in a subject with orthopedicassociated muscle degeneration an effective amount of a compositioncomprising decellularized extracellular matrix derived from skeletalmuscle tissue.
 12. The method of claim 11, where said composition iscoated on an implant.
 13. The method of claim 11, wherein saidcomposition is delivered as a liquid or a powder.
 14. The method ofclaim 13, wherein said composition degrades within one to three monthsfollowing injection or implantation.
 15. The method of claim 11, whereininjection or implantation of said composition repairs skeletal muscletissue damage caused by acute sarcomere hyperelongation and myofibrillardisruption in said subject.
 16. The method of claim 13, wherein saidcomposition transitions to a gel form after delivery.
 17. The method ofclaim 11, wherein said effective amount is an amount that increasesblood flow, increases muscle mass, or induces new vascular formation inthe area of the injection or implantation of the treated subject. 18.The method of claim 11, wherein the effective amount is effective fortreating at least one of the symptoms selected from the group consistingof dysfunction of striated muscles, which include the rotator cuffmuscles (supraspinatus, infraspinatus, teres minor, subcapularis), hipabductor muscle (gluteus medius, gluteus minimus, gluteus maximus, andshort external rotators), foot and ankle muscles (tibialis posterior,gastrocnemius, soleus), lumbar spine muscles (multifidus, erectorspinae), and knee extensor muscles (quadriceps).
 19. A pharmaceuticalcomposition for treating muscle degeneration in a subject comprising: atherapeutically effective amount of decellularized extracellular matrixderived from skeletal muscle tissue.
 20. The composition of claim 19,wherein the muscle tissue, from which the decellularized extracellularmatrix is derived, is tissue specific and selected to mimic thebiochemical cues, as well as the microenvironment of the native musclebeing treated for muscle degeneration.
 21. The composition of claim 20,wherein the native muscle being treated for muscle degenerationcomprises pelvic floor musculature.
 22. The composition of claim 20,wherein the tissue specific muscle from which the decellularizedextracellular matrix is derived is selected for treating at least one ofthe symptoms selected from the group consisting of dysfunction ofstriated muscles, which include the rotator cuff muscles (supraspinatus,infraspinatus, teres minor, subcapularis), hip abductor muscle (gluteusmedius, gluteus minimus, gluteus maximus, and short external rotators),foot and ankle muscles (tibialis posterior, gastrocnemius, soleus),lumbar spine muscles (multifidus, erector spinae), and knee extensormuscles (quadriceps).
 23. The composition of claim 20, wherein thetissue specific muscle from which the decellularized extracellularmatrix is derived is selected for treating at least one of the symptomsselected from the group consisting of pelvic floor muscle (PFM)fibrosis, pelvic floor disorders (PFD), urinary (UI) and fecalincontinence (FI), pelvic organ prolapse (POP), dysfunction of pelvicstriated muscles, external urethral (EUS) and external anal (EAS)sphincteric muscles, rectal prolapse (RP), stress urinary incontinence(SUI), and mixed urinary incontinence (MUI).